TGF-ß/Smad2/3 signaling directly regulates several miRNAs in mouse ES cells and early embryos.

The Transforming Growth Factor-ß (TGF-ß) signaling pathway is one of the major pathways essential for normal embryonic development and tissue homeostasis, with anti-tumor but also pro-metastatic properties in cancer. This pathway directly regulates several target genes that mediate its downstream functions, however very few microRNAs (miRNAs) have been identified as targets. miRNAs are modulators of gene expression with essential roles in development and a clear association with diseases including cancer. Little is known about the transcriptional regulation of the primary transcripts (pri-miRNA, pri-miR) from which several mature miRNAs are often derived. Here we present the identification of miRNAs regulated by TGF-ß signaling in mouse embryonic stem (ES) cells and early embryos. We used an inducible ES cell system to maintain high levels of the TGF-ß activated/phosphorylated Smad2/3 effectors, which are the transcription factors of the pathway, and a specific inhibitor that blocks their activation. By performing short RNA deep-sequencing after 12 hours Smad2/3 activation and after 16 hours inhibition, we generated a database of responsive miRNAs. Promoter/enhancer analysis of a subset of these miRNAs revealed that the transcription of pri-miR-181c/d and the pri-miR-341∼3072 cluster were found to depend on activated Smad2/3. Several of these miRNAs are expressed in early mouse embryos, when the pathway is known to play an essential role. Treatment of embryos with TGF-ß inhibitor caused a reduction of their levels confirming that they are targets of this pathway in vivo. Furthermore, we showed that pri-miR-341∼3072 transcription also depends on FoxH1, a known Smad2/3 transcription partner during early development. Together, our data show that miRNAs are regulated directly by the TGF-ß/Smad2/3 pathway in ES cells and early embryos. As somatic abnormalities in functions known to be regulated by the TGF-ß/Smad2/3 pathway underlie tumor suppression and metastasis, this research also provides a resource for miRNAs involved in cancer.

Two polypyrimidine tracts in the nitric oxide synthase 2 gene: similar regulatory sequences with different properties.

We reported previously that the polymorphic polypyrimidine CCTTT-microsatellite in the regulatory region of nitric oxide synthase 2 (NOS2) bound nuclear proteins in vitro. In the present work, we aimed to characterize and investigate a potential regulatory role of the CCTTT-microsatellite in NOS2 expression. Therefore, we performed gel-shift, S1-nuclease, and chromatin immunoprecipitation (ChIP) assays. In vitro experiments showed that the microsatellite formed triplex-DNA both with and without superhelical constraint. We also found that the CCTTT-microsatellite and an apparently similar CT-repeat in the first intron of NOS2 were specifically cleaved by S1-nuclease, when cloned into a supercoiled plasmid. In vitro data suggested that the CCTTT-microsatellite bound both polypyrimidine tract-binding protein (PTBP1) and heterogeneous nuclear ribonucleoprotein K (hnRNPK). On the contrary, ChIP revealed binding of PTBP1 and hnRNPK rather to the CT-repeat in the first intron than to the CCTTT-microsatellite. Enrichment for RNA polymerase II and acetylated histones H3 and H4 was also detected at the intronic site. We suggest that both PTBP1 and hnRNPK binds the single strand of the triplex-DNA formed at the CT-repeat in the first intron and that this interaction could be involved in the regulation of NOS2 expression.

Differential binding and co-binding pattern of FOXA1 and FOXA3 and their relation to H3K4me3 in HepG2 cells revealed by ChIP-seq.

BACKGROUND: The forkhead box/winged helix family members FOXA1, FOXA2, and FOXA3 are of high importance in development and specification of the hepatic linage and the continued expression of liver-specific genes. RESULTS: Here, we present a genome-wide location analysis of FOXA1 and FOXA3 binding sites in HepG2 cells through chromatin immunoprecipitation with detection by sequencing (ChIP-seq) studies and compare these with our previous results on FOXA2. We found that these factors often bind close to each other in different combinations and consecutive immunoprecipitation of chromatin for one and then a second factor (ChIP-reChIP) shows that this occurs in the same cell and on the same DNA molecule, suggestive of molecular interactions. Using co-immunoprecipitation, we further show that FOXA2 interacts with both FOXA1 and FOXA3 in vivo, while FOXA1 and FOXA3 do not appear to interact. Additionally, we detected diverse patterns of trimethylation of lysine 4 on histone H3 (H3K4me3) at transcriptional start sites and directionality of this modification at FOXA binding sites. Using the sequence reads at polymorphic positions, we were able to predict allele specific binding for FOXA1, FOXA3, and H3K4me3. Finally, several SNPs associated with diseases and quantitative traits were located in the enriched regions. CONCLUSIONS: We find that ChIP-seq can be used not only to create gene regulatory maps but also to predict molecular interactions and to inform on the mechanisms for common quantitative variation.

Novel genes in cell cycle control and lipid metabolism with dynamically regulated binding sites for sterol regulatory element-binding protein 1 and RNA polymerase II in HepG2 cells detected by chromatin immunoprecipitation with microarray detection.

Sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and SREBP-2) are important regulators of genes involved in cholesterol and fatty acid metabolism, but have also been implicated in the regulation of the cell cycle and have been associated with the pathogenesis of type 2 diabetes, atherosclerosis and obesity, among others. In this study, we aimed to characterize the binding sites of SREBP-1 and RNA polymerase II through chromatin immunoprecipitation and microarray analysis in 1% of the human genome, as defined by the Encyclopaedia of DNA Elements consortium, in a hepatocellular carcinoma cell line (HepG2). Our data identified novel binding sites for SREBP-1 in genes directly or indirectly involved in cholesterol metabolism, e.g. apolipoprotein C-III (APOC3). The most interesting biological findings were the binding sites for SREBP-1 in genes for host cell factor C1 (HCFC1), involved in cell cycle regulation, and for filamin A (FLNA). For RNA polymerase II, we found binding sites at classical promoters, but also in intergenic and intragenic regions. Furthermore, we found evidence of sterol-regulated binding of SREBP-1 and RNA polymerase II to HCFC1 and FLNA. From the results of this work, we infer that SREBP-1 may be involved in processes other than lipid metabolism.