The hypoxia-inducible factors (HIFs) 1alpha and 2alpha are key mammalian transcription factors that exhibit dramatic increases in both protein stability and intrinsic transcriptional potency during low-oxygen stress. This increased stability is due to the absence of proline hydroxylation, which in normoxia promotes binding of HIF to the von Hippel-Lindau (VHL tumor suppressor) ubiquitin ligase. We now show that hypoxic induction of the COOH-terminal transactivation domain (CAD) of HIF occurs through abrogation of hydroxylation of a conserved asparagine in the CAD. Inhibitors of Fe(II)- and 2-oxoglutarate-dependent dioxygenases prevented hydroxylation of the Asn, thus allowing the CAD to interact with the p300 transcription coactivator. Replacement of the conserved Asn by Ala resulted in constitutive p300 interaction and strong transcriptional activity. Full induction of HIF-1alpha and -2alpha, therefore, relies on the abrogation of both Pro and Asn hydroxylation, which during normoxia occur at the degradation and COOH-terminal transactivation domains, respectively.

Mammalian cells adapt to hypoxic conditions through a transcriptional response pathway mediated by the hypoxia-inducible factor, HIF. HIF transcriptional activity is suppressed under normoxic conditions by hydroxylation of an asparagine residue within its C-terminal transactivation domain, blocking association with coactivators.Here we show that the protein FIH-1, previously shown to interact with HIF, is an asparaginyl hydroxylase. Like known hydroxylase enzymes, FIH-1 is an Fe(II)-dependent enzyme that uses molecular O 2 to modify its substrate. Together with the recently discovered prolyl hydroxylases that regulate HIF stability, this class of oxygen-dependent enzymes comprises critical regulatory components of the hypoxic response pathway. Received March 14, 2002; revised version accepted April 30, 2002. Almost all mammalian cells possess the ability to recognize changes in the local availability of oxygen. When oxygen levels are low (hypoxia), a conserved hypoxic response pathway is activated. At the center of this pathway lies the ubiquitously expressed transcription factor hypoxia-inducible factor (HIF) (Semenza 1999). HIF is a heterodimer composed of an alpha subunit, HIF-1␣ or the paralogs HIF-2␣ or HIF-3␣ (Tian et al. 1997;Gu et al. 1998;O'Rourke et al. 1999;Srinivas et al. 1999), and the HIF-1␤ subunit, also known as the aryl hydrocarbon receptor nuclear translocator (ARNT) (Wang et al. 1995). Whereas HIF-1␤ expression and activity levels remain largely unaffected by changes in oxygen levels, the HIF-␣ subunit is strongly induced following exposure to hypoxic conditions. Two primary mechanisms by which HIF-␣ activity is regulated by oxygen have been identified. Under normoxic conditions, the oxygen-dependent degradation domain (ODD) within the HIF-␣ subunit is recognized by the product of the von-Hippel Lindau tumor suppressor gene (pVHL) (Maxwell et al. 1999). pVHL is a component of a protein-ubiquitin ligase complex that targets the alpha subunit for degradation by the proteasome (Maxwell et al. 1999;Cockman et al. 2000;Ohh et al. 2000;Tanimoto et al. 2000). pVHL recognition of HIF-␣ is dependent on hydroxylation of proline residues within the ODD (Ivan et al. 2001;Jaakkola et al. 2001;Yu et al. 2001). Under hypoxic conditions, prolyl hydroxylation is blocked, resulting in increased HIF-␣ stability and accumulation (Ivan et al. 2001;Jaakkola et al. 2001;Yu et al. 2001). This posttranslational modification is carried out by a family of prolyl hydroxylase enzymes that bear structural and functional similarities to previously characterized hydroxylases (Bruick and McKnight 2001;Epstein et al. 2001). Like these enzymes, the HIF prolyl hydroxylase enzymes use Fe(II) to bind O 2 to hydroxylate both 2-oxoglutarate and the target proline residue (Bruick and McKnight 2001;Epstein et al. 2001). Because these enzymes bind oxygen directly, it has been speculated that they may be critical oxygen sensors within the hypoxic response pathway.In addition to inducing HIF stability, hypoxic conditions promote the ...

The folding of genomic DNA from the beads-on-a-string like structure of nucleosomes into higher order assemblies is critically linked to nuclear processes. We have calculated the first 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. This has allowed us to study genome folding down to a scale of <100 kb and to validate the structures. We show that the structures of individual topological-associated domains and loops vary very substantially from cell-to-cell. By contrast, A/B compartments, lamin-associated domains and active enhancers/promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. Through studying pluripotency factor- and NuRD-regulated genes, we illustrate how single cell genome structure determination provides a novel approach for investigating biological processes.

The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.

AlkB homolog 1 (ALKBH1) is one of nine members of the family of mammalian AlkB homologs. Most Alkbh1−/− mice die during embryonic development, and survivors are characterized by defects in tissues originating from the ectodermal lineage. In this study, we show that deletion of Alkbh1 prolonged the expression of pluripotency markers in embryonic stem cells and delayed the induction of genes involved in early differentiation. In vitro differentiation to neural progenitor cells (NPCs) displayed an increased rate of apoptosis in the Alkbh1−/− NPCs when compared with wild-type cells. Whole-genome expression analysis and chromatin immunoprecipitation revealed that ALKBH1 regulates both directly and indirectly, a subset of genes required for neural development. Furthermore, our in vitro enzyme activity assays demonstrate that ALKBH1 is a histone dioxygenase that acts specifically on histone H2A. Mass spectrometric analysis demonstrated that histone H2A from Alkbh1−/− mice are improperly methylated. Our results suggest that ALKBH1 is involved in neural development by modifying the methylation status of histone H2A. Stem Cells 2012;30:2672–2682

Hypoxia-inducible factor 1␣ (HIF-1␣) and the HIF-like factor (HLF) are two highly related basic Helix-LoopHelix/Per-Arnt-Sim (bHLH/PAS) homology transcription factors that undergo dramatically increased function at low oxygen levels. Despite strong similarities in their activation mechanisms (e.g. they both undergo rapid hypoxia-induced protein stabilization, bind identical target DNA sequences, and induce synthetic reporter genes to similar degrees), they are both essential for embryo survival via distinct functions during vascularization (HIF-1␣) or catecholamine production (HLF). It is currently unknown how such specificity of action is achieved. We report here that DNA binding by HLF, but not by HIF-1␣, is dependent upon reducing redox conditions. In vitro DNA binding and mammalian two-hybrid assays showed that a unique cysteine in the DNA-binding basic region of HLF is a target for the reducing activity of redox factor Ref- Cells of aerobic organisms depend upon a plentiful supply of oxygen for energy production, and consequently, a number of mechanisms have evolved to sense and respond to disruptions of oxygen homeostasis. Although several signal transduction and gene regulatory mechanisms have been established to operate in response to oxidative stresses induced by intracellular oxygen radical production or cell penetration by oxidants, mammalian gene regulatory pathways that respond to hypoxia (low oxygen) have only recently begun to be understood. Analysis of hypoxia-induced activation of the erythropoietin gene led to the cloning of HIF-1␣ 1 (1), a transcription factor that exhibits marked increases in stability and transcription potency at low intracellular oxygen levels (for a recent review, see Ref.2). HIF-1␣ is a member of a bHLH protein subfamily that contains PAS dimerization domains juxtaposed with the bHLH. A number of bHLH/PAS factors have now been cloned and found to have crucial roles in physiological functions as diverse as xenobiotic metabolism (the dioxin or Ah receptor), maintenance of circadian rhythms (Clock and Period), and neurogenesis (Single-minded) (for a recent review, see Ref.3). Like the dioxin receptor, HIF-1␣ responds to environmental cues to form a functional heterodimer with a general bHLH/ PAS partner protein, Arnt (Ah receptor nuclear translocator). A closely related factor, the HIF-like factor (HLF (4); also known as EPAS1 (endothelial PAS1) (5), HIF-related factor (6), and MOP2 (member of PAS family 2) (7)), shares 48% amino acid identity with HIF-1␣ and is correspondingly induced by hypoxia to form a transcriptionally active HLF/Arnt heterodimer. In vitro mechanistic analysis of HIF-1␣ and HLF revealed that they dimerize with Arnt with similar affinities (4); bind to the same consensus hypoxia response elements found in genes such as erythropoietin (EPO) and vascular endothelial growth factor; and in transient transfection assays, activate reporter genes with similar intensities when under hypoxic stress (4,5,8). RNA and protein blot analyses showed both HLF and HIF-1␣...

To sustain life mammals have an absolute and continual requirement for oxygen, which is necessary to produce energy for normal cell survival and growth. Hence, maintaining oxygen homeostasis is a critical requirement and mammals have evolved a wide range of cellular and physiological responses to adapt to changes in oxygen availability. In the past few years it has become evident that the transcriptional protein complex hypoxia-inducible factor (HIF) is a key regulator of these processes. In this review we will focus on the way oxygen availability regulates HIF proteins and in particular we will discuss the way oxygendependent hydroxylation of specific amino acid residues has been demonstrated to regulate HIF function at the level of both protein stability and transcriptional potency.

The nucleosome remodeling deacetylase (NuRD) complex is a highly conserved regulator of chromatin structure and transcription. Structural studies have shed light on this and other chromatin modifying machines, but much less is known about how they assemble and whether stable and functional sub-modules exist that retain enzymatic activity. Purification of the endogenous Drosophila NuRD complex shows that it consists of a stable core of subunits, while others, in particular the chromatin remodeler CHD4, associate transiently. To dissect the assembly and activity of NuRD, we systematically produced all possible combinations of different components using the MultiBac system, and determined their activity and biophysical properties. We carried out single-molecule imaging of CHD4 in live mouse embryonic stem cells, in the presence and absence of one of core components (MBD3), to show how the core deacetylase and chromatin-remodeling sub-modules associate in vivo. Our experiments suggest a pathway for the assembly of NuRD via preformed and active sub-modules. These retain enzymatic activity and are present in both the nucleus and the cytosol, an outcome with important implications for understanding NuRD function.