Transcription profiling by array of bone marrow derived macrophages from LyzCre/Trim33+/+ mice and LyzCre/Trim33flox/flox mice to study the role of TRIM33 in the transcriptional response induced by pathogen receptors
ABSTRACT: Macrophage activation during the innate immune response is tightly regulated to prevent tissue damage while activating the defense to cellular attack. Using a mouse model where Trim33 is specifically deleted in mature myeloid cells, we show that TRIM33 is essential for two aspects of the inflammatory response in vivo. Loss of TRIM33 attenuates the initiation of macrophage activation by lipopolysaccharide (LPS) and TRIM33 is necessary to switch off transcription of inflammatory genes during late stages of LPS activation. Using chromatin immunoprecipitation coupled to deep sequencing, we provide a link between TRIM33 binding, RNA Polymerase II occupancy and H3K4me3 spreading on inflammatory genes in macrophages and reveal novel insights concerning the transcriptional regulation of Ifn-beta where TRIM33 exerts a repressive function via a distal regulatory region during late stages of LPS activation of macrophages. These findings pinpoint TRIM33 as a major regulator of the resolution of inflammation and indicate that transcriptional regulators can fine-tune H3K4me3 spreading. To study the role of TRIM33 in the transcriptional response induced by pathogen receptors, we analyzed whether lack of TRIM33 in macrophages affected the TLR-mediated regulation of proinflammatory and antimicrobial genes. To study this role, we bred TRIM33fl/fl mice with Lyz-Cre mice (obtained from The Jackson Laboratory, Bar Harbor, Maine, USA) where the Cre recombinase gene is under the regulatory sequences of the Lyz gene that is expressed only in mature myeloid cells. Bone marrow cells from 2 LyzCre/Trim33+/+ mice and 2 LyzCre/Trim33flox/flox mice were then differentiated in macrophages and treated during 0h, 4h, 12h and 24h with LPS. Total RNA was extracted from macrophages and analysed using cDNA microarrays. The set of gene expression consists of 16 samples of RNA of bone marrow derived macrophages activated with 100ng/ml of LPS during 0h, 4h, 12h, 24h, 8 samples from 2 LyzCre/Trim33+/+ mice and 8 samples from 2 LyzCre/Trim33flox/flox mice.
Project description:The CpG island methylator phenotype (CIMP) in colorectal tumors can be recognized by an increased frequency of aberrant methylation in a specific set of genomic loci. Because of the strong association of CIMP with high microsatellite instability (MSI-H), the identification of CIMP+ tumors within microsatellite stable (MSS) colorectal cancers may not be straightforward. To overcome this potential limitation, we have built an improved 7-loci set of methylation markers that includes CACNA1G, IGF2, RUNX3, HTR6, RIZ1, MINT31 and MAP1B. This new set of CIMP markers revealed a bimodal distribution of methylation frequencies in a group of 95 MSS colorectal cancers, which allowed a clearer separation between CIMP classes. Correlation of CIMP+ tumors with bio-pathological traits revealed significant associations with location to the proximal colon, mucinous histology and chromosomal stability. Although not statistically significant, a trend toward an adverse prognosis for CIMP+ cases was observed. Microarray analysis revealed that CIMP+ tumors are characterized by a unique expression profile, a result that confirms that CIMP+ tumors represent a distinct molecular class within MSS colorectal cancers. Moreover, our results suggest that this expression pattern may represent the molecular background for the development of CIMP+ tumors that, in turn, develop MSI when aberrant methylation occurs at the MLH1 gene promoter.
Project description:We investigate the relevance of RNA integrity in gene expression analysis as well as analysis methods to accommodate the possible effects of degradation using paired tumour and normal samples from colorectal cancer patients undergoing colonic resection.
Project description:In most mammalian species, a critical step of placental development is the fusion of trophoblast cell into a multinucleated syncytiotrophoblast layer, which separates the fetal and maternal blood circulations. Advancement in understanding this process came from the identification of two pairs of envelope genes of retroviral origin, independently acquired by the human (syncytin-1 and 2) and mouse (syncytin-A and B) genomes, specifically expressed in the placenta and with in vitro cell-cell fusion activity. We previously showed that syncytin-A is involved in the formation of one of the two syncytiotrophoblast layer of the mouse placenta -ST-I , facing the maternal lacuna- with syncytin-A null embryos dying at mid-gestation, and their placenta disclosing impaired formation of the ST-I layer. Here by generating syncytin-B KO mice, we demonstrated that syncytin-B null placenta have defects in formation of the syncytiotrophoblast layer-II facing the fetal blood vessels, with refined electron microscopy analyses showing evidence of unfused apposed cells. Lack of trophoblast fusion is associated with signs of trophoblast degeneration and with formation of huge maternal blood lacuna, ultimately disrupting the architecture of the labyrinth layer. These alterations did not result in complete abortion of syncytin-B null embryos, at variance with the syncytin-A KO phenotype, but in a reduced proportion of homozygous syncytin-B knockout individuals at birth, with evidence of late-onset embryonic growth retardation. Double knockout mice experiments demonstrate a premature death of syncytin-A null embryos if syncytin-B is deleted, thus strongly suggesting cooperative involvement of both syncytiotrophoblast layer-I and layer-II for the structural and functional integrity of the maternofetal interface and exchanges. Finally, a microarray analysis of wild-type and syncytin-B null placenta transcripts disclosed alterations in gene expression level for only a very limited number of genes, with the largest change (a 7-fold induction in the syncytin-B null placenta) observed for the connexin-30 gene. Immunohistochemistry further localized the connexin-30 protein in the labyrinth zone of mutant placenta, at the level of the fetomaternal interface. It is proposed that this might correspond to a compensatory process whereby disruption of syncytialization in syncytin-B knockout placenta is counteracted by an enhanced, gap junction mediated, cell-cell communication. These data demonstrate that syncytin-B is required for ST-II syncytial differentiation and the functional integrity of the labyrinth. Altogether, these findings definitively demonstrate that the two endogenous retroviral syncytin-A and -B Env genes contribute independently to the formation of the two syncytiotrophoblast layers during placenta formation, and provide additional insights into the subtle mechanisms of syncytiotrophoblast differentiation in the mouse. Experimental design: Placenta RNA of embryos homozygous for the syncytin-B deletion (SynB-/-) were compared to placental RNA of wild-type embryos (SynB+/+) of the same litter. Two stages of gestation, day 12 and d14, were analyzed. At day 12, 3 litters were obtained; for two of them, the RNAs were analyzed individually, and for the third one, placenta RNAs of the wild-type genotype were pooled. At day 14, one litter was obtained; RNA samples with the same genotype were pooled
Project description:A recent study of the characteristics of coexisting melanoma and renal cell carcinoma RCC in the same patients supports a genetic predisposition underlying the association between these two cancers. The microphthalmia associated transcription factor (MITF) was proposed to act as a melanoma oncogene; it also stimulates the transcription of hypoxia inducible factor (HIF1A), whose pathway is targeted by kidney cancer susceptibility genes. By sequencing the MITF gene, we detected a germ line missense substitution (p.E318K) in 5 of 62 patients affected with melanoma and RCC. When compared with 1,659 controls, this MITF substitution occurred at a significantly higher frequency in melanoma + RCC patients (p = 1.3x10-4), melanoma -only patients (p = 7.8 x10-5), and RCC-only patients (p = 0.008). Overall, p.E318K substitution carriers had a higher than fivefold increased risk of developing melanoma, RCC or both cancers (odds-ratio = 5.55, 95% confidence interval = 2.59 to 12.91). Codon 318 falls in the second MITF-conserved, small-ubiquitin like modifier (SUMO)-1 consensus binding site (YKXE) and the p.E318K substitution severely impairs SUMOylation of MITF. MITF p.E318K binds more efficiently to the HIF1A promoter and increases its activation compared to the wild type MITF. In RCC cell line, transcriptomic approach identifies a MITF p.E318K signature related to cell growth, proliferation and inflammation. MITF p.E318K is more potent than wild type MITF in promoting melanocytic and renal cell clonogenicity, migration and invasion, consistent with a gain-of-function role in tumorigenesis. Our data provide novel insights on the link between transcription, sumoylation and cancer, possibly mediated by oxidative stress. This set of 26 arrays presents expression data for 3 conditions (reference cell line, with addition of wt MITF or e318K MITF for two different cell lines (melanoma A375 and renal RCC4), in dye swap and 3 independant biological replicates.
Project description:Docetaxel is used as a standard treatment in patients with metastatic castration-resistant prostate cancer. However, a large subset of patients develops resistance by mechanisms that remain largely unknown. It is thus important to define the relevant pathways implicated in docetaxel-resistance and validate predictive biomarkers that will allow approaches of personalized treatment. In this aim, we established resistant IGR-CaP1 prostate cancer cell lines to different doses of docetaxel (IGR-CaP1-R cell lines) and investigated gene expression profiles by microarray analyses. We generated a signature of 112 genes potentially implicated in docetaxel-resistance whose expression is highly modified (Fold change ≥ 5). Among these genes, significant modification of expression was observed among cell cycle components in the resistant cells. Hence, we focused on the role of the cell cycle regulator LZTS1 located on chromosome 8p which was under-expressed in all our docetaxel-resistant models. LZTS1 extinction was confirmed at the RNA and protein levels. DNA methylation analysis revealed a stretch of 20 highly methylated CpGs in the region encompassing the exon 1 of LZTS1 promoter in the docetaxel-resistant cells suggesting the existence of an epigenetic regulation of LZTS1 expression in the resistant cells. By using siRNA strategy, we found evidence that LZTS1 plays an important role in the acquisition of the resistant phenotype. In addition, immunohistochemical staining showed that LZTS1 protein was absent or down-regulated in 33% of diagnostic biopsies obtained in patients with metastatic castration-resistant prostate cancer. This heterogeneous labeling suggests that LZTS1 might constitute a predictive biomarker of response to docetaxel chemotherapy. Furthermore, as Cdc25C is a LZTS1 partner in the mitosis regulation, we observed that targeting of Cdc25C with the pharmacological Cdc25C inhibitor NSC 663284 specifically killed the docetaxel-resistant cells. These results strongly suggest that Cdc25C plays a role in docetaxel resistance and that Cdc25C might be a therapeutic target to overcome docetaxel resistance. Altogether our findings identify an important role of LZTS1 in developing docetaxel resistance in prostate cancer through its role in regulating phosphatase Cdc25C. The set of gene expression with 4x44K Agilent ( design 014850) correspond to 6 doses of docetaxel 2?5 to 200 ug/ml) in dual color and dye-swap versus the IGR-Cap1 cell line without docetaxel.