Genome-Wide Association Study Identifies 4 Novel Risk Loci for Small Intestinal Neuroendocrine Tumors Including a Missense Mutation in LGR5.

BACKGROUND & AIMS: Small intestinal neuroendocrine tumor (SI-NET) is a rare disease, but its incidence has increased over the past 4 decades. Understanding the genetic risk factors underlying SI-NETs can help in disease prevention and may provide clinically beneficial markers for diagnosis. Here the results of the largest genome-wide association study of SI-NETs performed to date with 405 cases and 614,666 controls are reported. METHODS: Samples from 307 patients with SI-NETs and 287,137 controls in the FinnGen study were used for the identification of SI-NET risk-associated genetic variants. The results were also meta-analyzed with summary statistics from the UK Biobank (n = 98 patients with SI-NET and n = 327,529 controls). RESULTS: We identified 6 genome-wide significant (P < 5 × 10-8) loci associated with SI-NET risk, of which 4 (near SEMA6A, LGR5, CDKAL1, and FERMT2) are novel and 2 (near LTA4H-ELK and in KIF16B) have been reported previously. Interestingly, the top hit (rs200138614; P = 1.80 × 10-19) was a missense variant (p.Cys712Phe) in the LGR5 gene, a bona-fide marker of adult intestinal stem cells and a potentiator of canonical WNT signaling. The association was validated in an independent Finnish collection of 70 patients with SI-NETs, as well as in the UK Biobank exome sequence data (n = 92 cases and n = 392,814 controls). Overexpression of LGR5 p.Cys712Phe in intestinal organoids abolished the ability of R-Spondin1 to support organoid growth, indicating that the mutation perturbed R-Spondin-LGR5 signaling. CONCLUSIONS: Our study is the largest genome-wide association study to date on SI-NETs and reported 4 new associated genome-wide association study loci, including a novel missense mutation (rs200138614, p.Cys712Phe) in LGR5, a canonical marker of adult intestinal stem cells.

Tellu - an object-detector algorithm for automatic classification of intestinal organoids.

Intestinal epithelial organoids recapitulate many of the in vivo features of the intestinal epithelium, thus representing excellent research models. Morphology of the organoids based on light-microscopy images is used as a proxy to assess the biological state of the intestinal epithelium. Currently, organoid classification is manual and, therefore, subjective and time consuming, hampering large-scale quantitative analyses. Here, we describe Tellu, an object-detector algorithm trained to classify cultured intestinal organoids. Tellu was trained by manual annotation of >20,000 intestinal organoids to identify cystic non-budding organoids, early organoids, late organoids and spheroids. Tellu can also be used to quantify the relative organoid size, and can classify intestinal organoids into these four subclasses with accuracy comparable to that of trained scientists but is significantly faster and without bias. Tellu is provided as an open, user-friendly online tool to benefit the increasing number of investigations using organoids through fast and unbiased organoid morphology and size analysis.

Inactivation of AMPK Leads to Attenuation of Antigen Presentation and Immune Evasion in Lung Adenocarcinoma.

PURPOSE: Mutations in STK11 (LKB1) occur in 17% of lung adenocarcinoma (LUAD) and drive a suppressive (cold) tumor immune microenvironment (TIME) and resistance to immunotherapy. The mechanisms underpinning the establishment and maintenance of a cold TIME in LKB1-mutant LUAD remain poorly understood. In this study, we investigated the role of the LKB1 substrate AMPK in immune evasion in human non-small cell lung cancer (NSCLC) and mouse models and explored the mechanisms involved. EXPERIMENTAL DESIGN: We addressed the role of AMPK in immune evasion in NSCLC by correlating AMPK phosphorylation and immune-suppressive signatures and by deleting AMPKα1 (Prkaa1) and AMPKα2 (Prkaa2) in a KrasG12D -driven LUAD. Furthermore, we dissected the molecular mechanisms involved in immune evasion by comparing gene-expression signatures, AMPK activity, and immune infiltration in mouse and human LUAD and gain or loss-of-function experiments with LKB1- or AMPK-deficient cell lines. RESULTS: Inactivation of both AMPKα1 and AMPKα2 together with Kras activation accelerated tumorigenesis and led to tumors with reduced infiltration of CD8+/CD4+ T cells and gene signatures associated with a suppressive TIME. These signatures recapitulate those in Lkb1-deleted murine LUAD and in LKB1-deficient human NSCLC. Interestingly, a similar signature is noted in human NSCLC with low AMPK activity. In mechanistic studies, we find that compromised LKB1 and AMPK activity leads to attenuated antigen presentation in both LUAD mouse models and human NSCLC. CONCLUSIONS: The results provide evidence that the immune evasion noted in LKB1-inactivated lung cancer is due to subsequent inactivation of AMPK and attenuation of antigen presentation.

Ciliary Hedgehog signaling patterns the digestive system to generate mechanical forces driving elongation.

How tubular organs elongate is poorly understood. We found that attenuated ciliary Hedgehog signaling in the gut wall impaired patterning of the circumferential smooth muscle and inhibited proliferation and elongation of developing intestine and esophagus. Similarly, ablation of gut-wall smooth muscle cells reduced lengthening. Disruption of ciliary Hedgehog signaling or removal of smooth muscle reduced residual stress within the gut wall and decreased activity of the mechanotransductive effector YAP. Removing YAP in the mesenchyme also reduced proliferation and elongation, but without affecting smooth muscle formation, suggesting that YAP interprets the smooth muscle-generated force to promote longitudinal growth. Additionally, we developed an intestinal culture system that recapitulates the requirements for cilia and mechanical forces in elongation. Pharmacologically activating YAP in this system restored elongation of cilia-deficient intestines. Thus, our results reveal that ciliary Hedgehog signaling patterns the circumferential smooth muscle to generate radial mechanical forces that activate YAP and elongate the gut.

LKB1 Represses ATOH1 via PDK4 and Energy Metabolism and Regulates Intestinal Stem Cell Fate.

BACKGROUND & AIMS: In addition to the Notch and Wnt signaling pathways, energy metabolism also regulates intestinal stem cell (ISC) function. Tumor suppressor and kinase STK11 (also called LKB1) regulates stem cells and cell metabolism. We investigated whether loss of LKB1 alters ISC homeostasis in mice. METHODS: We deleted LKB1 from ISCs in mice using Lgr5-regulated CRE-ERT2 (Lkb1Lgr5-KO mice) and the traced lineages by using a CRE-dependent TdTomato reporter. Intestinal tissues were collected and analyzed by immunohistochemical and immunofluorescence analyses. We purified ISCs and intestinal progenitors using flow cytometry and performed RNA-sequencing analysis. We measured organoid-forming capacity and ISC percentages using intestinal tissues from Lkb1Lgr5-KO mice. We analyzed human Ls174t cells with knockdown of LKB1 or other proteins by immunoblotting, real-time quantitative polymerase chain reaction, and the Seahorse live-cell metabolic assay. RESULTS: Some intestinal crypts from Lkb1Lgr5-KO mice lost ISCs compared with crypts from control mice. However, most crypts from Lkb1Lgr5-KO mice contained functional ISCs that expressed increased levels of Atoh1 messenger RNA (mRNA), acquired a gene expression signature associated with secretory cells, and generated more cells in the secretory lineage compared with control mice. Knockdown of LKB1 in Ls174t cells induced expression of Atoh1 mRNA and a phenotype of increased mucin production; knockdown of ATOH1 prevented induction of this phenotype. The increased expression of Atoh1 mRNA after LKB1 loss from ISCs or Ls174t cells did not involve Notch or Wnt signaling. Knockdown of pyruvate dehydrogenase kinase 4 (PDK4) or inhibition with dichloroacetate reduced the up-regulation of Atoh1 mRNA after LKB1 knockdown in Ls174t cells. Cells with LKB1 knockdown had a reduced rate of oxygen consumption, which was partially restored by PDK4 inhibition with dichloroacetate. ISCs with knockout of LKB1 increased the expression of PDK4 and had an altered metabolic profile. CONCLUSIONS: LKB1 represses transcription of ATOH1, via PDK4, in ISCs, restricting their differentiation into secretory lineages. These findings provide a connection between metabolism and the fate determination of ISCs.

Increased α-actinin-1 destabilizes E-cadherin-based adhesions and associates with poor prognosis in basal-like breast cancer.

The controlled formation and stabilization of E-cadherin-based adhesions is vital for epithelial integrity. This requires co-operation between the E-cadherin-based adhesions and the associated actin cytoskeleton. In cancer, this co-operation often fails, predisposing cells to migration through molecular mechanisms that have only been partially characterized. Here, we demonstrate that the actin filament cross-linker α-actinin-1 is frequently increased in human breast cancer. In mammary epithelial cells, the increased α-actinin-1 levels promote cell migration and induce disorganized acini-like structures in Matrigel. This is accompanied by a major reorganization of the actin cytoskeleton and the associated E-cadherin-based adhesions. Increased expression of α-actinin-1 is particularly noted in basal-like breast cancer cell lines, and in breast cancer patients it associates with poor prognosis in basal-like subtypes. Downregulation of α-actinin-1 in E-cadherin expressing basal-like breast cancer cells demonstrate that α-actinin-1-assembled actin fibers destabilize E-cadherin-based adhesions. Taken together, these results indicate that increased α-actinin-1 expression destabilizes E-cadherin-based adhesions, which is likely to promote the migratory potential of breast cancer cells. Furthermore, our results identify α-actinin-1 as a candidate prognostic biomarker in basal-like breast cancer.

Depletion of Mediator Kinase Module Subunits Represses Superenhancer-Associated Genes in Colon Cancer Cells.

In cancer, oncogene activation is partly mediated by acquired superenhancers, which therefore represent potential targets for inhibition. Superenhancers are enriched for BRD4 and Mediator, and both BRD4 and the Mediator MED12 subunit are disproportionally required for expression of superenhancer-associated genes in stem cells. Here we show that depletion of Mediator kinase module subunit MED12 or MED13 together with MED13L can be used to reduce expression of cancer-acquired superenhancer genes, such as the MYC gene, in colon cancer cells, with a concomitant decrease in proliferation. Whereas depletion of MED12 or MED13/MED13L caused a disproportional decrease of superenhancer gene expression, this was not seen with depletion of the kinases cyclin-dependent kinase 9 (CDK8) and CDK19. MED12-MED13/MED13L-dependent superenhancer genes were coregulated by ß-catenin, which has previously been shown to associate with MED12. Importantly, ß-catenin depletion caused reduced binding of MED12 at the MYC superenhancer. The effect of MED12 or MED13/MED13L depletion on cancer-acquired superenhancer gene expression was more specific than and partially distinct from that of BRD4 depletion, with the most efficient inhibition seen with combined targeting. These results identify a requirement of MED12 and MED13/MED13L for expression of acquired superenhancer genes in colon cancer, implicating these Mediator subunits as potential therapeutic targets for colon cancer, alone or together with BRD4.

Stromal Lkb1 deficiency leads to gastrointestinal tumorigenesis involving the IL-11-JAK/STAT3 pathway.

Germline mutations in the gene encoding tumor suppressor kinase LKB1 lead to gastrointestinal tumorigenesis in Peutz-Jeghers syndrome (PJS) patients and mouse models; however, the cell types and signaling pathways underlying tumor formation are unknown. Here, we demonstrated that mesenchymal progenitor- or stromal fibroblast-specific deletion of Lkb1 results in fully penetrant polyposis in mice. Lineage tracing and immunohistochemical analyses revealed clonal expansion of Lkb1-deficient myofibroblast-like cell foci in the tumor stroma. Loss of Lkb1 in stromal cells was associated with induction of an inflammatory program including IL-11 production and activation of the JAK/STAT3 pathway in tumor epithelia concomitant with proliferation. Importantly, treatment of LKB1-defcient mice with the JAK1/2 inhibitor ruxolitinib dramatically decreased polyposis. These data indicate that IL-11-mediated induction of JAK/STAT3 is critical in gastrointestinal tumorigenesis following Lkb1 mutations and suggest that targeting this pathway has therapeutic potential in Peutz-Jeghers syndrome.

AMPK negatively regulates tensin-dependent integrin activity.

Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin-ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a ß1-integrin inhibitor in fibroblasts. Loss of AMPK promotes ß1-integrin activity, the formation of centrally located active ß1-integrin- and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases ß1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind ß1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes.

SUMOylation of AMPKα1 by PIAS4 specifically regulates mTORC1 signalling.

AMP-activated protein kinase (AMPK) inhibits several anabolic pathways such as fatty acid and protein synthesis, and identification of AMPK substrate specificity would be useful to understand its role in particular cellular processes and develop strategies to modulate AMPK activity in a substrate-specific manner. Here we show that SUMOylation of AMPKα1 attenuates AMPK activation specifically towards mTORC1 signalling. SUMOylation is also important for rapid inactivation of AMPK, to allow prompt restoration of mTORC1 signalling. PIAS4 and its SUMO E3 ligase activity are specifically required for the AMPKα1 SUMOylation and the inhibition of AMPKα1 activity towards mTORC1 signalling. The activity of a SUMOylation-deficient AMPKα1 mutant is higher than the wild type towards mTORC1 signalling when reconstituted in AMPKα-deficient cells. PIAS4 depletion reduced growth of breast cancer cells, specifically when combined with direct AMPK activator A769662, suggesting that inhibiting AMPKα1 SUMOylation can be explored to modulate AMPK activation and thereby suppress cancer cell growth.

Cyclin-dependent kinase 8 module expression profiling reveals requirement of mediator subunits 12 and 13 for transcription of Serpent-dependent innate immunity genes in Drosophila.

The Cdk8 (cyclin-dependent kinase 8) module of Mediator integrates regulatory cues from transcription factors to RNA polymerase II. It consists of four subunits where Med12 and Med13 link Cdk8 and cyclin C (CycC) to core Mediator. Here we have investigated the contributions of the Cdk8 module subunits to transcriptional regulation using RNA interference in Drosophila cells. Genome-wide expression profiling demonstrated separation of Cdk8-CycC and Med12-Med13 profiles. However, transcriptional regulation by Cdk8-CycC was dependent on Med12-Med13. This observation also revealed that Cdk8-CycC and Med12-Med13 often have opposite transcriptional effects. Interestingly, Med12 and Med13 profiles overlapped significantly with that of the GATA factor Serpent. Accordingly, mutational analyses indicated that GATA sites are required for Med12-Med13 regulation of Serpent-dependent genes. Med12 and Med13 were also found to be required for Serpent-activated innate immunity genes in defense to bacterial infection. The results reveal a novel role for the Cdk8 module in Serpent-dependent transcription and innate immunity.

N-methylnitrosourea aggravates gastrointestinal polyposis in Lkb1+/- mice.

Peutz-Jeghers patients develop hamartomatous polyps and carcinomas of the gastrointestinal tract. Cyclooxygenase-2 accelerates polyp growth in Lkb1 (+/-) mice modelling Peutz-Jeghers polyposis. In this study, we aimed to evaluate the effect of the mutagenic carcinogen N-methylnitrosourea (MNU) on gastrointestinal tumourigenesis in Lkb1 (+/-) mice and to investigate the role of cyclooxygenase-2 on the tumourigenesis. We treated 40 Lkb1 (+/-) and 51 wild-type mice with MNU, 10 mice from both groups received the cyclooxygenase-2 inhibitor celecoxib. Carcinogen-treated Lkb1 (+/-) mice displayed worse survival (60%) than treated wild-type (100%, P = 0.028) or untreated Lkb1 (+/-) mice (92%, P = 0.045). Also, the gastrointestinal tumour burden was almost 10-fold higher in carcinogen-treated (2181 mm(3)) than in untreated (237 mm(3), P = 0.00045) Lkb1 (+/-) mice. Celecoxib was much less efficient in reducing tumourigenesis in MNU-treated mice (by 23%; 1686 mm(3)) than in untreated mice (76%; 58 mm(3)). Surprisingly, the increase in tumour burden in MNU-treated mice was not accompanied by consistent histological changes, with only a single focus of epithelial dysplasia noted. This study suggests that MNU promotes Peutz-Jeghers polyposis independently from the acceleration by cyclooxygenase-2.

CCRK depletion inhibits glioblastoma cell proliferation in a cilium-dependent manner.

Loss of primary cilia is frequently observed in tumour cells, including glioblastoma cells, and proposed to benefit tumour growth, but a causal link has not been established. Here, we show that CCRK (cell cycle-related kinase) and its substrate ICK (intestinal cell kinase) inhibit ciliogenesis. Depletion of CCRK leads to accumulation of ICK at ciliary tips, altered ciliary transport and inhibition of cell cycle re-entry in NIH3T3 fibroblasts. In glioblastoma cells with deregulated high levels of CCRK, its depletion restores cilia through ICK and an ICK-related kinase MAK, thereby inhibiting glioblastoma cell proliferation. These results indicate that inhibition of ciliogenesis might be a mechanism used by cancer cells to provide a growth advantage.

Assembly of non-contractile dorsal stress fibers requires α-actinin-1 and Rac1 in migrating and spreading cells.

Cell migration and spreading is driven by actin polymerization and actin stress fibers. Actin stress fibers are considered to contain α-actinin crosslinkers and nonmuscle myosin II motors. Although several actin stress fiber subtypes have been identified in migrating and spreading cells, the degree of molecular diversity of their composition and the signaling pathways regulating fiber subtypes remain largely uncharacterized. In the present study we identify that dorsal stress fiber assembly requires α-actinin-1. Loss of dorsal stress fibers in α-actinin-1-depleted cells results in defective maturation of leading edge focal adhesions. This is accompanied by a delay in early cell spreading and slower cell migration without noticeable alterations in myosin light chain phosphorylation. In agreement with the unaltered myosin II activity, dorsal stress fiber trunks lack myosin II and are resistant to myosin II ATPase inhibition. Furthermore, the non-contractility of dorsal stress fibers is supported by the finding that Rac1 induces dorsal stress fiber assembly whereas contractile ventral stress fibers are induced by RhoA. Loss of dorsal stress fibers either by depleting α-actinin-1 or Rac1 results in a ß-actin accumulation at the leading edge in migrating and spreading cells. These findings molecularly specify dorsal stress fibers from other actin stress fiber subtypes. Furthermore, we propose that non-contractile dorsal stress fibers promote cell migration and early cell spreading through Rac1-induced actin polymerization.

Tumor suppressor function of Liver kinase B1 (Lkb1) is linked to regulation of epithelial integrity.

Although loss of epithelial integrity is a hallmark of advanced cancer, it remains poorly understood whether genetic alterations corrupting this integrity causally facilitate tumorigenesis. We show that conditional deletion of tumor suppressor gene Lkb1 (Par-4) in the mammary gland compromises epithelial integrity manifested by mislocalization of cell polarity markers, lateralization of tight junctions, deterioration of desmosomes and basement membrane (BM), and hyperbranching of the mammary ductal tree. We identify the desmosomal BM remodelling serine protease Hepsin as a key factor mediating Lkb1 loss-induced structural alterations in mammary epithelium and BM fragmentation. Although loss of Lkb1 alone does not promote mammary tumorigenesis, combination of Lkb1 deficiency with oncogenic c-Myc leads to dramatic acceleration in tumor formation. The results coupling Lkb1 loss-mediated epithelial integrity defects to mislocalization of serine protease Hepsin and to oncogenic synergy with c-Myc imply that Lkb1 loss facilitates oncogenic proliferation by releasing epithelial cells from structural BM boundaries.

Inhibition of cyclooxygenase-2 causes regression of gastric adenomas in trefoil factor 1 deficient mice.

Cyclooxygenase-2 (Cox-2) expression is a marker of reduced survival in gastric cancer patients, and inhibition of Cox-2 suppresses gastrointestinal carcinogenesis in experimental animal models. To investigate the role of Cox-2 in gastric carcinogenesis in vivo, we utilized trefoil factor 1 (Tff1) deficient mice, which model the neoplastic process of the stomach by developing gastric adenomas with full penetrance. These tumors express Cox-2 protein and mRNA, and we have now investigated the effects of genetic deletion of the mouse Cox-2 gene [also known as prostaglandin-endoperoxide synthase 2 (Ptgs2)] and a Cox-2 selective drug celecoxib. Our results show that genetic deletion of Cox-2 in the Tff1 deleted background resulted in reduced adenoma size and ulceration with a chronic inflammatory reaction at the site of the adenoma. To characterize the effect of Cox-2 inhibition in more detail, mice that had already developed an adenoma were fed with celecoxib for 8-14 weeks, which resulted in disruption of the adenoma that ranged from superficial erosion to deep ulcerated destruction accompanied with chronic inflammation. Importantly, mice fed with celecoxib for 16 weeks, followed by control food for 9 weeks, redeveloped a complete adenoma with no detectable inflammatory process. Finally, we determined the identity of the Cox-2 expressing cells and found them to be fibroblasts. Our results show that inhibition of Cox-2 is sufficient to reversibly disrupt gastric adenomas in mice.

The tumor suppressor kinase LKB1: lessons from mouse models.

Mutations in the tumor suppressor gene LKB1 are important in hereditary Peutz-Jeghers syndrome, as well as in sporadic cancers including lung and cervical cancer. LKB1 is a kinase-activating kinase, and a number of LKB1-dependent phosphorylation cascades regulate fundamental cellular and organismal processes in at least metabolism, polarity, cytoskeleton organization, and proliferation. Conditional targeting approaches are beginning to demonstrate the relevance and specificity of these signaling pathways in development and homeostasis of multiple organs. More than one of the pathways also appear to contribute to tumor growth following Lkb1 deficiencies based on a number of mouse tumor models. Lkb1-dependent activation of AMPK and subsequent inactivation of mammalian target of rapamycin signaling are implicated in several of the models, and other less well characterized pathways are also involved. Conditional targeting studies of Lkb1 also point an important role of LKB1 in epithelial-mesenchymal interactions, significantly expanding knowledge on the relevance of LKB1 in human disease.

Phosphorylated ß-catenin localizes to centrosomes of neuronal progenitors and is required for cell polarity and neurogenesis in developing midbrain.

ß-catenin has well-established functions in cell growth and differentiation as part of the Wnt signaling pathway and in regulation of cellular adhesion with E-cadherin. Here we studied its significance in midbrain development by temporally controlled deletion of ß-catenin allowing simultaneous analysis of complete (ß-cat-null) and partial (ß-cat-low) loss-of-function phenotypes in progenitor cells. ß-cat-null cells did not contain centrosomes or a microtubule network and were unpolarized forming delaminated bulges. ß-cat-low cells displayed defects in the orientation of the mitotic spindle, increased asymmetric cell divisions and premature differentiation in absence of alterations in polarity or adhesion. The spindle defect was associated with decreased centrosomal S33/S34/T41 phosphorylated ß-catenin (p-ß-cat) and centrosomal and microtubule defects. Interestingly, neural progenitor cells in mice expressing only unphosphorylatable ß-catenin share several phenotypes with ß-catenin loss-of-function mice with defects in microtubules and polarity. The results demonstrate a novel function for p-ß-cat in maintaining neuroepithelial integrity and suggest that centrosomal p-ß-cat is required to maintain symmetric cleavages and polarity in neural progenitors.

LKB1 signaling in advancing cell differentiation.

The Peutz-Jeghers syndrome (PJS) culprit kinase LKB1 phosphorylates and activates multiple intracellular kinases regulating cell metabolism and polarity. The relevance of each of these pathways is highly variable depending on the tissue type, but typically represents functions of differentiated cells. These include formation and maintenance of specialized cell compartments in nerve axons, swift refunneling of metabolites and restructuring of cell architecture in response to environmental cues in committed lymphocytes, and ensuring energy-efficient oxygen-based energy expenditure. Such features are often lost or reduced in cancer cells, and indeed LKB1 defects in PJS-associated and sporadic cancers and even the benign PJS polyps lead to differentiation defects, including expansion of partially differentiated epithelial cells in PJS polyps and epithelial-to-mesenchymal transition in carcinomas. This review focuses on the involvement of LKB1 in the differentiation of epithelial, mesenchymal, hematopoietic and germinal lineages.

Requirement of TFIIH kinase subunit Mat1 for RNA Pol II C-terminal domain Ser5 phosphorylation, transcription and mRNA turnover.

The relevance of serine 5 phosphorylation of RNA polymerase II carboxy-terminal domain during initiation has been difficult to determine in mammalian cells as no general in vivo Ser5 kinase has been identified. Here, we demonstrate that deletion of the TFIIH kinase subunit Mat1 in mouse fibroblasts leads to dramatically reduced Pol II Ser5 phosphorylation. This is associated with defective capping and reduced Ser2 phosphorylation, decreased Pol II progression into elongation and severely attenuated transcription detected through analysis of nascent mRNAs, establishing a general requirement for mammalian Mat1 in transcription. Surprisingly, the general defect in Pol II transcription in Mat1(-/-) fibroblasts is not reflected in the majority of steady-state mRNAs. This indicates widespread stabilization of mRNAs and points to the existence of a regulatory mechanism to stabilize mRNAs following transcriptional attenuation, thus revealing a potential caveat in similar studies limited to analysis of steady-state mRNAs.