Molecular interactions between HNF4a, FOXA2 and GABP identified at regulatory DNA elements through ChIP-sequencing

Gene expression is regulated by combinations of transcription factors, which can be mapped to regulatory elements on a genome-wide scale using ChIP experiments. In a previous ChIP-chip study of USF1 and USF2 we found evidence also of binding of GABP, FOXA2 and HNF4a within the enriched regions. Here, we have applied ChIP-seq for these transcription factors and identified 3064 peaks of enrichment for GABP, 7266 for FOXA2 and 18783 for HNF4a. Distal elements with USF2 signal was frequently bound also by HNF4a and FOXA2. GABP peaks were found at transcription start sites, whereas 94% of FOXA2 and 90% of HNF4a peaks were located at other positions. We developed a method to accurately define TFBS within peaks, and found the predicted sites to have an elevated conservation level compared to peak centers; however the majority of bindings were not evolutionary conserved. An interaction between HNF4a and GABP was seen at TSS, with one-third of the HNF4a positive promoters being bound also by GABP, and this interaction was verified by co-immunoprecipitations

Genetic and Genomic Analysis of Transcriptional Regulation in Human Cells

There are around 20.000 genes in the human genome all of which could potentially be expressed. However, it is obvious that not all of them can be active at the same time. Thus, there is a need for coordination achieved through the regulation of transcription. Transcriptional regulation is a crucial multi-component process involving genetic and epigenetic factors, which determine when and how genes are expressed. The aim of this thesis was to study two of these components, the transcription factors and the DNA sequence elements with which they interact. In papers I and II, we tried to characterize the regulatory role of repeated elements in the regulatory sequences of nitric oxide synthase 2 gene. We found that this type of repeat is able to adopt non B-DNA conformations in vitro and that it binds nuclear factors, in addition to RNA polymerase II. Therefore it is probable that these types of repeats can participate in the regulation of genes. In papers III-V, we intended to analyze the genome-wide binding sites for six transcription factors involved in fatty acid and cholesterol metabolism and the sites of an epigenetic mark in a human liver cell line. For this, we applied the chromatin immunoprecipitation (ChIP) method together with detection on microarrays (ChIP-chip) or by detection with the new generation massively parallel sequencers (ChIP-seq). We found that all of these transcription factors are involved in other liver-specific processes than metabolism, for example cell proliferation. We were also able to define two sets of transcription factors depending on the position of their binding relative to gene promoters. Finally, we demonstrated that the patterns of the epigenetic mark reflect the structure and transcriptional activity of the promoters. In conclusion, this thesis presents experiments, which moves our view from genetics to genomics, from in vitro to in vivo, and from low resolution to high resolution analysis of transcriptional regulation

Genetic and Genomic Analysis of Transcriptional Regulation in Human Cells

There are around 20.000 genes in the human genome all of which could potentially be expressed. However, it is obvious that not all of them can be active at the same time. Thus, there is a need for coordination achieved through the regulation of transcription. Transcriptional regulation is a crucial multi-component process involving genetic and epigenetic factors, which determine when and how genes are expressed. The aim of this thesis was to study two of these components, the transcription factors and the DNA sequence elements with which they interact. In papers I and II, we tried to characterize the regulatory role of repeated elements in the regulatory sequences of nitric oxide synthase 2 gene. We found that this type of repeat is able to adopt non B-DNA conformations in vitro and that it binds nuclear factors, in addition to RNA polymerase II. Therefore it is probable that these types of repeats can participate in the regulation of genes. In papers III-V, we intended to analyze the genome-wide binding sites for six transcription factors involved in fatty acid and cholesterol metabolism and the sites of an epigenetic mark in a human liver cell line. For this, we applied the chromatin immunoprecipitation (ChIP) method together with detection on microarrays (ChIP-chip) or by detection with the new generation massively parallel sequencers (ChIP-seq). We found that all of these transcription factors are involved in other liver-specific processes than metabolism, for example cell proliferation. We were also able to define two sets of transcription factors depending on the position of their binding relative to gene promoters. Finally, we demonstrated that the patterns of the epigenetic mark reflect the structure and transcriptional activity of the promoters. In conclusion, this thesis presents experiments, which moves our view from genetics to genomics, from in vitro to in vivo, and from low resolution to high resolution analysis of transcriptional regulation

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

miRNA target predictions for negatively regulated p-Smad2/3 target genes.

<p>– No predicted miRNA target sites.</p

Expression of p-Smad2/3 inducible miRNAs in the early embryo.

<p>(<b>A</b>) qPCR was performed on RNA from pooled mouse E6.5 embryos to detect the relative expression levels of the p-Smad2/3 inducible miRNAs. Error bars show ±SEM of triplicate assays. ‘^†’ denotes the direct p-Smad2/3 target miRNAs. (<b>B–F</b>) <i>In situ</i> hybridizations were performed on E6.5 embryos using Locked Nucleic Acid modified oligonucleotides against miRs-382-5p (<b>B, E</b>), -181d-5p (<b>C, F</b>) and -499-5p (negative control) (<b>D</b>). Images are representative of 32 embryos for -382-5p, 29 for -181d-5p, and 22 for -499-5p as indicated in the images (n). Dotted lines in (<b>B, C</b>) indicate positions of sections presented in (<b>E, F</b>), respectively. Scale bars for whole mounts indicate 100 µm (<b>B, C, D</b>), and for sections 50 µm (<b>E, F</b>). Extra-embryonic (Exe), epiblast (Ep) and endoderm (En).</p

Expression profile of negatively regulated p-Smad2/3 target genes in TAG1 ES cells.

<p>Analysis of Affymetrix expression microarray on RNA extracted from TAG1 ES cells either pre-treated for 6 hrs with SB-431542 (SB) followed by 15 hrs with doxycycline (Dox), and 6 hrs with SB (<b>A</b>) or pre-treated for 6 hrs with SB followed by 12 hrs with Dox, and 9 hrs with SB (<b>B</b>). Genes with >1.2-fold decrease in expression after 15 hrs Dox treatment (15 hrs vs. 0 hr) and >1.2-fold increase after 6 hrs SB treatment (21 hrs vs. 15 hrs) (<b>A</b>) and after 12 hrs Dox (12 hrs vs. 0 hr) and 9 hrs SB treatment (21 hrs vs. 12 hrs) (<b>B</b>) are presented in the graph. All time points were normalized to the 0 hr non-induced control. Several genes were measured by multiple probe sets as indicated.xl.</p

Identification of p-Smad2/3 inducible miRNAs.

<p>(<b>A</b>) Western blot analysis of p-Smad2 and PCNA (loading control) levels in TAG1 ES cells untreated (0), treated with SB-431542 (SB) for 16 hrs (16) or with SB for 16 hrs followed by doxycycline (Dox) for 12 hrs (16+12). (<b>B</b>) Relative fold induction of mature miRNAs in TAG1 cells treated as in (<b>A</b>) detected by small RNA deep-sequencing. (<b>C</b>) qPCR validation of the deep-sequencing data, normalized to the non-Smad2/3 responsive miR-26a. Error bars represent ±SEM of triplicate reactions.</p

Pri-miRNA genes encoding the p-Smad2/3-inducible miRNAs.

†<p>Directly regulated p-Smad2/3-responsive miRNAs.</p