Following the realization that eukaryotic RNA-binding proteomes are substantially larger than anticipated, we must now understand their detailed composition and dynamics. Methods such as RNA interactome capture (RIC) have begun to address this need. However, limitations of RIC have been reported. Here we describe enhanced RNA interactome capture (eRIC), a method based on the use of an LNA-modified capture probe, which yields numerous advantages including greater specificity and increased signal-to-noise ratios compared to existing methods. In Jurkat cells, eRIC reduces the rRNA and DNA contamination by >10-fold compared to RIC and increases the detection of RNA-binding proteins. Due to its low background, eRIC also empowers comparative analyses of changes of RNA-bound proteomes missed by RIC. For example, in cells treated with dimethyloxalylglycine, which inhibits RNA demethylases, eRIC identifies m6A-responsive RNA-binding proteins that escape RIC. eRIC will facilitate the unbiased characterization of RBP dynamics in response to biological and pharmacological cues.

SUMMARY Hypoxia inducible factors (HIFs) are critical regulators of the cellular response to hypoxia. Despite their established roles in normal physiology and numerous pathologies, the molecular mechanisms by which they control gene expression remain poorly understood. We report here a conserved role for the TIP60 complex as a HIF1 transcriptional cofactor in Drosophila and human cells. TIP60 (KAT5) is required for HIF1-dependent gene expression in fly cells and embryos, and colorectal cancer cells. HIF1A interacts with and recruits TIP60 to chromatin. TIP60 is dispensable for HIF1A association with its target genes but is required for HIF1A-dependent chromatin modification and RNA polymerase II activation in hypoxia. In human cells, global analysis of HIF1A-dependent gene activity reveals that most HIF1A targets require either TIP60, the CDK8-Mediator complex, or both as co-activators for full expression in hypoxia. Thus, HIF1A employs functionally diverse cofactors to regulate different subsets of genes within its transcriptional program.

Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF–dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF–related pathologies, including heart attack, cancer, and stroke.

Reduction in oxygen levels below normal concentrations plays important roles in different normal and pathological conditions, such as development, tumorigenesis, chronic kidney disease and stroke. Organisms exposed to hypoxia trigger changes at both cellular and systemic levels to recover oxygen homeostasis. Most of these processes are mediated by Hypoxia Inducible Factors, HIFs, a family of transcription factors that directly induce the expression of several hundred genes in mammalian cells. Although different aspects of HIF regulation are well known, it is still unclear by which precise mechanism HIFs activate transcription of their target genes. Concomitantly, hypoxia provokes a dramatic decrease of general transcription that seems to rely in part on epigenetic changes through a poorly understood mechanism. In this review we discuss the current knowledge on chromatin changes involved in HIF dependent gene activation, as well as on other epigenetic changes, not necessarily linked to HIF that take place under hypoxic conditions.