PITX1, a specificity determinant in the HIF-1α-mediated transcriptional response to hypoxia

Hypoxia is an important developmental cue for multicellular organisms but it is also a contributing factor for several human pathologies, such as stroke, cardiovascular diseases and cancer. In cells, hypoxia activates a major transcriptional program coordinated by the Hypoxia Inducible Factor (HIF) family. HIF can activate more than one hundred targets but not all of them are activated at the same time, and there is considerable cell type variability. In this report we identified the paired-like homeodomain pituitary transcription factor (PITX1), as a transcription factor that helps promote specificity in HIF-1α dependent target gene activation. Mechanistically, PITX1 associates with HIF-1β and it is important for the induction of certain HIF-1 dependent genes but not all. In particular, PITX1 controls the HIF-1α-dependent expression of the histone demethylases; JMJD2B, JMJD2A, JMJD2C and JMJD1B. Functionally, PITX1 is required for the survival and proliferation responses in hypoxia, as PITX1 depleted cells have higher levels of apoptotic markers and reduced proliferation. Overall, our study identified PITX1 as a key specificity factor in HIF-1α dependent responses, suggesting PITX1 as a protein to target in hypoxic cancers

ABHD11 maintains 2-oxoglutarate metabolism by preserving functional lipoylation of the 2-oxoglutarate dehydrogenase complex

Abstract: 2-oxoglutarate (2-OG or α-ketoglutarate) relates mitochondrial metabolism to cell function by modulating the activity of 2-OG dependent dioxygenases involved in the hypoxia response and DNA/histone modifications. However, metabolic pathways that regulate these oxygen and 2-OG sensitive enzymes remain poorly understood. Here, using CRISPR Cas9 genome-wide mutagenesis to screen for genetic determinants of 2-OG levels, we uncover a redox sensitive mitochondrial lipoylation pathway, dependent on the mitochondrial hydrolase ABHD11, that signals changes in mitochondrial 2-OG metabolism to 2-OG dependent dioxygenase function. ABHD11 loss or inhibition drives a rapid increase in 2-OG levels by impairing lipoylation of the 2-OG dehydrogenase complex (OGDHc)—the rate limiting step for mitochondrial 2-OG metabolism. Rather than facilitating lipoate conjugation, ABHD11 associates with the OGDHc and maintains catalytic activity of lipoyl domain by preventing the formation of lipoyl adducts, highlighting ABHD11 as a regulator of functional lipoylation and 2-OG metabolism

Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins. Using multiplex 47snoMEM vectors that co-express multiple 47snoMEN in a single transcript, each of which can target different sites in the same mRNA, we document >3-fold increase in knock-down efficiency when compared with the original HBII-180C based snoMEN. The multiplex 47snoMEM vector allowed the construction of human protein replacement cell lines with improved efficiency, including the establishment of novel GFP–HIF-1α replacement cells. Quantitative mass spectrometry analysis confirmed the enhanced efficiency and specificity of protein replacement using the 47snoMEN-PR vectors. The 47snoMEN vectors expand the potential applications for snoMEN technology in gene expression studies, target validation and gene therapy

Tapasin's protein interactions in the rainbow trout peptide-loading complex

The final publication is available at Elsevier via http://dx.doi.org/10.1016/j.dci.2017.12.015 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Major histocompatibility complex (MHC) class I receptors play a key role in the immune system by presenting non-self peptides to T cell lymphocytes. In humans, the assembly of the MHC class I with a peptide is mediated by machinery in the endoplasmic reticulum referred as the peptide loading complex (PLC). Although, the identity of the PLC has been widely explored in humans, this complex has not been characterized in fish. Co-immunoprecipitation and mass spectrometry analysis revealed that the protein-protein interactions which exist in the human PLC are conserved in the monocyte/macrophage rainbow trout cell line (RTS11), in particular the interaction of tapasin with the transporter associated with antigen processing (TAP), MHC class I and ERp57. Importantly, a 20 kDa tapasin version that contains an intact C and N terminal domains was found to associate with ERp57 and form a 75 kDa heterodimer. These results suggest a possible novel alternative spliced version of tapasin may regulate the formation of the peptide-loading complex in teleosts.NSERC Discovery Grant number 217529-2008Canada Research Council Research Chair held by B

Functional Characterization of Transcription Factor Motifs Using Cross-species Comparison across Large Evolutionary Distances

We address the problem of finding statistically significant associations between cis-regulatory motifs and functional gene sets, in order to understand the biological roles of transcription factors. We develop a computational framework for this task, whose features include a new statistical score for motif scanning, the use of different scores for predicting targets of different motifs, and new ways to deal with redundancies among significant motif–function associations. This framework is applied to the recently sequenced genome of the jewel wasp, Nasonia vitripennis, making use of the existing knowledge of motifs and gene annotations in another insect genome, that of the fruitfly. The framework uses cross-species comparison to improve the specificity of its predictions, and does so without relying upon non-coding sequence alignment. It is therefore well suited for comparative genomics across large evolutionary divergences, where existing alignment-based methods are not applicable. We also apply the framework to find motifs associated with socially regulated gene sets in the honeybee, Apis mellifera, using comparisons with Nasonia, a solitary species, to identify honeybee-specific associations

Genetic approaches to understand cellular responses to oxygen availability

Funder: Lister Institute of Preventive Medicine; Id: http://dx.doi.org/10.13039/501100001255Funder: NIHR Cambridge Biomedical Research CentreFunder: Addenbrooke's Charitable TrustOxygen‐sensing mechanisms have evolved to allow organisms to respond and adapt to oxygen availability. In metazoans, oxygen‐sensing is predominantly mediated by the hypoxia inducible factors (HIFs). These transcription factors are stabilised when oxygen is limiting, activating genes involved in angiogenesis, cell growth, pH regulation and metabolism to reset cell function and adapt to the cellular environment. However, the recognition that other cellular pathways and enzymes can also respond to changes in oxygen abundance provides further complexity. Dissecting this interplay of oxygen‐sensing mechanisms has been a key research goal. Here, we review how genetic approaches have contributed to our knowledge of oxygen‐sensing pathways which to date have been predominantly focused on the HIF pathway. We discuss how genetic studies have advanced the field and outline the implications and limitations of such approaches for the development of therapies targeting oxygen‐sensing mechanisms in human disease