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Surgical resection is the mainstay in intended curative treatment of colorectal cancer (CRC) and may be accompanied by adjuvant chemotherapy. However, 40% of the patients experience recurrence within five years of treatment, highlighting the importance of improved, personalized treatment options. Monolayer cell cultures and murine models, which are generally used to study the biology of CRC, are associated with certain drawbacks; hence, the use of organoids has been emerging. Organoids obtained from tumors display similar genotypic and phenotypic characteristics, making them ideal for investigating individualized treatment strategies and for integration as a core platform to be used in prediction models. Here, we review studies correlating the clinical response in patients with CRC with the therapeutic response in patient-derived organoids (PDO), as well as the limitations and potentials of this model. The studies outlined in this review reported strong associations between treatment responses in the PDO model and clinical treatment responses. However, as PDOs lack the tumor microenvironment, they do not genuinely account for certain crucial characteristics that influence therapeutic response. To this end, we reviewed studies investigating PDOs co-cultured with tumor-infiltrating lymphocytes. This model is a promising method allowing evaluation of patient-specific tumors and selection of personalized therapies. Standardized methodologies must be implemented to reach a “gold standard” for validating the use of this model in larger cohorts of patients. The introduction of this approach to a clinical scenario directing neoadjuvant treatment and in other curative and palliative treatment strategies holds incredible potential for improving personalized treatment and its outcomes.

Dysregulation of interleukin 33 (IL-33) has been implicated in the pathogenesis of several autoimmune and inflammatory diseases, but few studies have examined transcriptional regulation of the IL33 gene. In the intestines, gene regulation is controlled by a transcription factor network of which the intestinal-specific transcription factor CDX2 is a key component. In this study, we investigated whether CDX2 regulates IL33 mRNA expression. We examined IL33 mRNA expression in primary colonic epithelial cells from healthy humans and epithelial cell lines, revealing high expression levels in primary colonic and LS174T cells. Combining genomics data (ChIP-Seq, RNA-Seq) and IL33 promoter analyses in LS174T cells revealed intronic enhancer activity in the IL33 gene that is dependent on CDX2 expression. Western blotting and qRT-PCR confirmed that IL33 expression is upregulated in a CDX2 concentration-dependent manner, thereby providing the first evidence that CDX2 regulates the expression of IL33.

Sequencing of primary colorectal tumors has identified a gene fusion in approximately 3% of colorectal cancer patients of the VTI1A and TCF7L2 genes, encoding a VTI1A-TCF4 fusion protein containing a truncated TCF4. As dysregulation of the Wnt signaling pathway is associated with colorectal cancer development and progression, the functional properties and transcriptional regulation of the VTI1A-TCF4 fusion protein may also play a role in these processes. Functional characteristics of the VTI1A-TCF4 fusion protein in Wnt signaling were analyzed in NCI-H508 and LS174T colon cancer cell lines. The NCI-H508 cell line, containing the VTI1A-TCF7L2 fusion gene, showed no active Wnt signaling, and overexpression of the VTI1A-TCF4 fusion protein in LS174T cells along with a Wnt signaling luciferase reporter plasmid showed inhibition of activity. The transcriptional regulation of the VTI1A-TCF4 fusion gene was investigated in LS174T cells where the activity of the VTI1A promoter was compared to that of the TCF7L2 promoter, and the transcription factor CDX2 was analyzed for gene regulatory activity of the VTI1A promoter through luciferase reporter gene assay using colon cancer cell lines as a model. Transfection of LS174T cells showed that the VTI1A promoter is highly active compared to the TCF7L2 promoter, and that CDX2 activates transcription of VTI1A. These results suggest that the VTI1A-TCF4 fusion protein is a dominant negative regulator of the Wnt signaling pathway, and that transcription of VTI1A is activated by CDX2.

Inflammatory bowel disease (IBD) is a chronic intestinal disorder, with two main types: Crohn's disease (CD) and ulcerative colitis (UC), whose molecular pathology is not well understood. The majority of IBD-associated SNPs are located in non-coding regions and are hard to characterize since regulatory regions in IBD are not known. Here we profile transcription start sites (TSSs) and enhancers in the descending colon of 94 IBD patients and controls. IBD-upregulated promoters and enhancers are highly enriched for IBD-associated SNPs and are bound by the same transcription factors. IBD-specific TSSs are associated to genes with roles in both inflammatory cascades and gut epithelia while TSSs distinguishing UC and CD are associated to gut epithelia functions. We find that as few as 35 TSSs can distinguish active CD, UC, and controls with 85% accuracy in an independent cohort. Our data constitute a foundation for understanding the molecular pathology, gene regulation, and genetics of IBD.

Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential genes that require absence of endogenous gene expression.

Upon developmental or environmental cues, the composition of transcription factors in a transcriptional regulatory network is deeply implicated in controlling the signature of the gene expression and thereby specifies the cell or tissue type. Novel methods including ChIP-chip and ChIP-Seq have been applied to analyze known transcription factors and their interacting regulatory DNA elements in the intestine. The intestine is an example of a dynamic tissue where stem cells in the crypt proliferate and undergo a differentiation process toward the villus. During this differentiation process, specific regulatory networks of transcription factors are activated to target specific genes, which determine the intestinal cell fate. The expanding genomewide mapping of transcription factor binding sites and construction of transcriptional regulatory networks provide new insight into how intestinal differentiation occurs. This review summarizes the current overview of the transcriptional regulatory networks driving epithelial differentiation in adult intestine. The novel technologies that have been implied to study these networks are presented and their prospects for implications in future research are also addressed.

Analyzing data obtained from genome-wide gene expression experiments is challenging due to the quantity of variables, the need for multivariate analyses, and the demands of managing large amounts of data. Here we present the R package pcaGoPromoter, which facilitates the interpretation of genome-wide expression data and overcomes the aforementioned problems. In the first step, principal component analysis (PCA) is applied to survey any differences between experiments and possible groupings. The next step is the interpretation of the principal components with respect to both biological function and regulation by predicted transcription factor binding sites. The robustness of the results is evaluated using cross-validation, and illustrative plots of PCA scores and gene ontology terms are available. pcaGoPromoter works with any platform that uses gene symbols or Entrez IDs as probe identifiers. In addition, support for several popular Affymetrix GeneChip platforms is provided. To illustrate the features of the pcaGoPromoter package a serum stimulation experiment was performed and the genome-wide gene expression in the resulting samples was profiled using the Affymetrix Human Genome U133 Plus 2.0 chip. Array data were analyzed using pcaGoPromoter package tools, resulting in a clear separation of the experiments into three groups: controls, serum only and serum with inhibitor. Functional annotation of the axes in the PCA score plot showed the expected serum-promoted biological processes, e.g., cell cycle progression and the predicted involvement of expected transcription factors, including E2F. In addition, unexpected results, e.g., cholesterol synthesis in serum-depleted cells and NF-¿B activation in inhibitor treated cells, were noted. In summary, the pcaGoPromoter R package provides a collection of tools for analyzing gene expression data. These tools give an overview of the input data via PCA, functional interpretation by gene ontology terms (biological processes), a

Background/aims: High levels of pro-inflammatory cytokines are linked to inflammatory bowel disease (IBD). The transcription factor Caudal-related homeobox transcription factor 2 (CDX2) plays a crucial role in differentiation of intestinal epithelium and regulates IBD-susceptibility genes, including meprin 1A (MEP1A). The aim was to investigate the expression of CDX2 and MEP1A in colitis; to assess if they are regulated by tumor necrosis factor-a (TNF-a), and finally to reveal if CDX2 is involved in a TNF-a-induced down-regulation of MEP1A. Methods: Expression of CDX2 and MEP1A was investigated in colonic biopsies of ulcerative colitis (UC) patients and in dextran sodium sulfate (DSS)-induced colitis. CDX2 protein expression was investigated by immunoblotting and immunohistochemical procedures. CDX2 and MEP1A regulation was examined in TNF-a-treated Caco-2 cells by reverse transcription-polymerase chain reaction and with reporter gene assays, and the effect of anti-TNF-a treatment was assessed using infliximab. Finally, in vivo CDX2-DNA interactions were investigated by chromatin immunoprecipitation. Results: The CDX2 and MEP1A mRNA expression was significantly decreased in active UC patients and in DSS-colitis. Colonic biopsy specimens from active UC showed markedly decreased CDX2 staining. TNF-a treatment diminished the CDX2 and MEP1A mRNA levels, a decrease which, was counteracted by infliximab treatment. Reporter gene assays showed significantly reduced CDX2 and MEP1A activity upon TNF-a stimulation. Finally, TNF-a impaired the ability of CDX2 to interact and activate its own, as well as the MEP1A expression. Conclusions: The present results indicate that a TNF-a-mediated down-regulation of CDX2 can be related to suppressed expression of MEP1A during intestinal inflammation.

Den humane population kan opdeles i to hovedgrupper baseret på deres ekspression af laktase som voksne. Laktase persistente udtrykker laktase hele livet hvorimod hos laktase non-persistente falder laktase ekspression til et lavt niveau ved 2-10 års alderen. Laktase non-persistens medfører ofte at laktose ikke bliver fordøjet effektivt og hvilket medfører laktose intolerans. Den laktase persistente fænotype er især almindelig i Nordeuropa og hos udvandrere fra denne region, men den findes også hyppigt i nogle afrikanske og arabiske populationer, f.eks hos Tuareg, Fulbe, Beja-folkene og beduiner. Målet for forskningsprojektet har været at afdække de molekylære mekanismer bag LPH ekspression både rummeligt og udviklingsmæssigt. Menneskets og grisens laktase promotere blev derfor klonet og analyseret funktionelt for dels at undersøge mekanismerne bag laktase persistens og non-persistens, men også for at belyse de fundamentale mekanismer bag den celle-specifikke ekspression af laktase i tyndtarmens enterocytter. Flere centrale gen-regulatoriske faktorer (Cdx-2, HNF1a, Fox, HNF4a, GATA-faktorer) med betydning for tarm-ekspression er blevet identificeret. Dette har givet et detaljeret billede af hvordan gen-regulatoriske DNA sekvenser kombineres for at opnå tarm-specific ekspression. Ved at studere den funktionelle betydning af en single nucleotide polymorphism, som er tæt associeret til laktase persistens/non-persistens, har vi identificeret transkriptionsfaktoren Oct-1. Oct-1 binder kraftigt til den laktase persistente SNP variant og denne binding er associeret med en kraftig gen-stimulerende aktivitet. Denne kraftige Oct-1 binding kan være hovedårsagen til, at laktase genet hos laktase persistente ikke nedreguleres. Forklaringen kan være, at Oct-1 kompenserer for en generel nedregulering af tarm-specifikke transkriptionsfaktorer efter diegivningsperioden. Denne generelle nedregulering forårsager, at laktase genet hos laktase non-persistente ikke bl, The human population can be divided in two main groups based on their intestinal expression of lactase as adult individuals. Lactase persistent individuals express lactase throughout their lifespan, whereas lactase non-persistent individuals lose their lactase expression between the ages of 2-10 years. Lactase non-persistence is often associated with an inability to efficiently digest lactose, resulting in lactose intolerance. The lactase persistent phenotype is frequently found among northern Europeans and their descendants, but is also found among some African and Arabic populations (e.g., Tuareg, Fulbe, Beja, and Bedouin people). The aim of this project is to investigate the molecular mechanisms behind the spatial and temporal expression of LPH. The human and the pig lactase promoter have therefore been cloned and functionally analyzed in order to understand the mechanisms behind lactase persistence and non-persistence, but also to elucidate the fundamental mechanisms behind the strict cell-specific expression of lactase in the mature enterocyte of the small intestine. Several key gene regulatory factors (Cdx-2, HNF1a, Fox's, HNF4a, Oct-1 and GATA-factors) important for intestinal expression have been identified, and this has fostered a detailed insight into how gene regulatory elements can be combined in promoters in order to direct intestinal-specific expression. By studying the functional importance of a single nucleotide polymorphism that is tightly associated with lactase persistence/non-persistence, it was found that Oct-1 binds strongly to the lactase persistent variant of the SNP. The binding of Oct-1 is associated with strong stimulating gene-regulatory activity. The strong Oct-1 binding can be the key event that prevents the decline of lactase during childhood, resulting in the lactase persistent phenotype. A possible explanation for the lactase persistent phenotype is that the binding of Oct-1 compensates for a lower level of intestin

Udgivelsesdato: May 25, A common genetically determined polymorphism in the human population leads to two distinct phenotypes in adults, lactase persistence and adult-type hypolactasia (lactase non-persistence). All healthy newborn children express high levels of lactase and are able to digest large quantities of lactose, the main carbohydrate in milk. Individuals with adult-type hypolactasia lose their lactase expression before adulthood and consequently often become lactose intolerant with associated digestive problems (e.g. diarrhoea). In contrast, lactase persistent individuals have a lifelong lactase expression and are able to digest lactose as adults. Lactase persistence can be regarded as the mutant phenotype since other mammals down-regulate their lactase expression after weaning (the postweaning decline). This phenomenon does not occur in lactase persistent individuals. The regulation of lactase expression is mainly transcriptional and it is well established that adult-type hypolactasia is inherited in an autosomal recessive manner, whereas persistence is dominant. The recent findings of single nucleotide polymorphisms associated with lactase persistence have made it possible to study the potential mechanisms underlying adult-type hypolactasia. This work has led to the identification of gene-regulatory sequences located far from the lactase gene (LCT). The present review describes the recent advances in the understanding of the regulation of lactase expression and the possible mechanisms behind adult-type hypolactasia.

Biokemi, Molekylærbiologi

Genregulering