The core promoter of eukaryotic genes is the minimal DNA region that recruits the basal transcription machinery to direct efficient and accurate transcription initiation. The fraction of human and yeast genes that contain specific core promoter elements such as the TATA box and the initiator (INR) remains unclear and core promoter motifs specific for TATA-less genes remain to be identified. Here, we present genome-scale computational analyses indicating that ∼76% of human core promoters lack TATA-like elements, have a high GC content, and are enriched in Sp1 binding sites. We further identify two motifs -M3 (SCGGAAGY) and M22 (TGCGCANK) -that occur preferentially in human TATA-less core promoters. About 24% of human genes have a TATA-like element and their promoters are generally AT-rich; however, only ∼10% of these TATA-containing promoters have the canonical TATA box (TATAWAWR). In contrast, ∼46% of human core promoters contain the consensus INR (YYANWYY) and ∼30% are INR-containing TATA-less genes. Significantly, ∼46% of human promoters lack both TATA-like and consensus INR elements. Surprisingly, mammalian-type INR sequences are present -and tend to cluster -in the transcription start site (TSS) region of ∼40% of yeast core promoters and the frequency of specific core promoter types appears to be conserved in yeast and human genomes. Gene Ontology analyses reveal that TATA-less genes in humans, as in yeast, are frequently involved in basic "housekeeping" processes, while TATAcontaining genes are more often highly regulated, such as by biotic or stress stimuli. These results reveal unexpected similarities in the occurrence of specific core promoter types and in their associated biological processes in yeast and humans and point to novel vertebrate-specific DNA motifs that might play a selective role in TATA-independent transcription.

Hepatocyte nuclear factor 4 alpha (HNF4α), a member of the nuclear receptor superfamily, is essential for liver function and is linked to several diseases including diabetes, hemophilia, atherosclerosis, and hepatitis. Although many DNA response elements and target genes have been identified for HNF4α the complete repertoire of binding sites and target genes in the human genome is unknown. Here, we adapt protein binding microarrays (PBMs) to examine the DNA-binding characteristics of two HNF4α species (rat and human) and isoforms (HNF4α2 and HNF4α8) in a high-throughput fashion. We identified ~1400 new binding sequences and used this dataset to successfully train a Support Vector Machine (SVM)model that predicts an additional ~10,000 unique HNF4α-binding sequences; we also identify new rules for HNF4α DNA binding. We performed expression profiling of an HNF4α RNA interference knockdown in HepG2 cells and compared the results to a search of the promoters of all human genes with the PBM and SVM models, as well as published genome-wide location analysis. Using this integrated approach, we identified ~240 new direct HNF4α human target genes, including new functional categories of genes not typically associated with HNF4α, such as cell cycle, immune function, apoptosis, stress response, and other cancer-related genes. Conclusion We report the first use of PBMs with a full-length liver-enriched transcription factor and greatly expand the repertoire of HNF4α-binding sequences and target genes, thereby identifying new functions for HNF4α. We also establish a web-based tool, HNF4 Motif Finder, that can be used to identify potential HNF4α-binding sites in any sequence.

SUMMARY The epigenetic activator Mixed lineage leukemia 1 (Mll1) is paramount for embryonic development and hematopoiesis. Here we demonstrate that the long, non-coding RNA (lncRNA) Mistral (Mira) activates transcription of the homeotic genes Hoxa6 and Hoxa7 in mouse embryonic stem cells (mESC) by recruiting Mll1 to chromatin. The Mira gene is located in the spacer DNA region (SDR) separating Hoxa6 and Hoxa7, transcriptionally silent in mESCs, and activated by retinoic acid. Mira-mediated recruitment of Mll1 to the Mira gene triggers dynamic changes in chromosome conformation, culminating in activation of Hoxa6 and Hoxa7 transcription. Hoxa6 and Hoxa7 activate the expression of genes involved in germ layer specification during mESC differentiation in a cooperative and redundant fashion. Our results connect the lncRNA Mira with the recruitment of Mll1 to target genes and implicate lncRNAs in epigenetic activation of gene expression during vertebrate cell fate determination.

Orphan nuclear receptors have been instrumental in identifying novel signaling pathways and therapeutic targets. However, identification of ligands for these receptors has often been based on random compound screens or other biased approaches. As a result, it remains unclear in many cases if the reported ligands are the true endogenous ligands, – i.e., the ligand that is bound to the receptor in an unperturbed in vivo setting. Technical limitations have limited our ability to identify ligands based on this rigorous definition. The orphan receptor hepatocyte nuclear factor 4 α (HNF4α) is a key regulator of many metabolic pathways and linked to several diseases including diabetes, atherosclerosis, hemophilia and cancer. Here we utilize an affinity isolation/mass-spectrometry (AIMS) approach to demonstrate that HNF4α is selectively occupied by linoleic acid (LA, C18:2ω6) in mammalian cells and in the liver of fed mice. Receptor occupancy is dramatically reduced in the fasted state and in a receptor carrying a mutation derived from patients with Maturity Onset Diabetes of the Young 1 (MODY1). Interestingly, however, ligand occupancy does not appear to have a significant effect on HNF4α transcriptional activity, as evidenced by genome-wide expression profiling in cells derived from human colon. We also use AIMS to show that LA binding is reversible in intact cells, indicating that HNF4α could be a viable drug target. This study establishes a general method to identify true endogenous ligands for nuclear receptors (and other lipid binding proteins), independent of transcriptional function, and to track in vivo receptor occupancy under physiologically relevant conditions.

HNF4α has been implicated in colitis and colon cancer in humans but the role of the different HNF4α isoforms expressed from the two different promoters (P1 and P2) active in the colon is not clear. Here, we show that P1-HNF4α is expressed primarily in the differentiated compartment of the mouse colonic crypt and P2-HNF4α in the proliferative compartment. Exon swap mice that express only P1- or only P2-HNF4α have different colonic gene expression profiles, interacting proteins, cellular migration, ion transport and epithelial barrier function. The mice also exhibit altered susceptibilities to experimental colitis (DSS) and colitis-associated colon cancer (AOM+DSS). When P2-HNF4α-only mice (which have elevated levels of the cytokine resistin-like β, RELMβ, and are extremely sensitive to DSS) are crossed with Retnlb-/- mice, they are rescued from mortality. Furthermore, P2-HNF4α binds and preferentially activates the RELMβ promoter. In summary, HNF4α isoforms perform non-redundant functions in the colon under conditions of stress, underscoring the importance of tracking them both in colitis and colon cancer.DOI: http://dx.doi.org/10.7554/eLife.10903.001