Fixed Cell Immunofluorescence for Quantification of Hypoxia-Induced Changes in Histone Methylation.

Hypoxia and its signalling pathway play a key role in human physiology and a variety of diseases. Alterations in histone methylation coordinate transcriptional responses to hypoxia. Here, we detail a fixed cell immunofluorescence method for quantifying hypoxia-induced changes in histone methylation, exemplified by the measurement of H3K27me3.

Regulation of chromatin accessibility by hypoxia and HIF.

Reduced oxygen availability (hypoxia) can act as a signalling cue in physiological processes such as development, but also in pathological conditions such as cancer or ischaemic disease. As such, understanding how cells and organisms respond to hypoxia is of great importance. The family of transcription factors called Hypoxia Inducible Factors (HIFs) co-ordinate a transcriptional programme required for survival and adaptation to hypoxia. However, the effects of HIF on chromatin accessibility are currently unclear. Here, using genome wide mapping of chromatin accessibility via ATAC-seq, we find hypoxia induces loci specific changes in chromatin accessibility are enriched at a subset hypoxia transcriptionally responsive genes, agreeing with previous data using other models. We show for the first time that hypoxia inducible changes in chromatin accessibility across the genome are predominantly HIF dependent, rapidly reversible upon reoxygenation and partially mimicked by HIF-α stabilisation independent of molecular dioxygenase inhibition. This work demonstrates that HIF is central to chromatin accessibility alterations in hypoxia, and has implications for our understanding of gene expression regulation by hypoxia and HIF.

Use of ChIP-qPCR to Study the Crosstalk Between HIF and NF-κB Signaling in Hypoxia and Normoxia.

Hypoxia and inflammation are intensely connected in a functional crosstalk. Within this crosstalk, two major transcription factors take center stage: HIF and NF-κB. To investigate transcription factor function, an important aspect is its ability to bind DNA. The most appropriate method to study this property in cells is the use of chromatin immunoprecipitation followed by qPCR and/or next generation sequencing. This allows identification of potentially directly regulated genes as well as enhancer regions. Here we describe the ChIP-qPCR method in detail, including key aspects important for the success of the technique.

Role of Hypoxia in the Control of the Cell Cycle.

The cell cycle is an important cellular process whereby the cell attempts to replicate its genome in an error-free manner. As such, mechanisms must exist for the cell cycle to respond to stress signals such as those elicited by hypoxia or reduced oxygen availability. This review focuses on the role of transcriptional and post-transcriptional mechanisms initiated in hypoxia that interface with cell cycle control. In addition, we discuss how the cell cycle can alter the hypoxia response. Overall, the cellular response to hypoxia and the cell cycle are linked through a variety of mechanisms, allowing cells to respond to hypoxia in a manner that ensures survival and minimal errors throughout cell division.

Oxygen-sensing mechanisms in cells.

The importance of oxygen for the survival of multicellular and aerobic organisms is well established and documented. Over the years, increased knowledge of its use for bioenergetics has placed oxygen at the centre of research on mitochondria and ATP-generating processes. Understanding the molecular mechanisms governing cellular oxygen sensing and response has allowed for the discovery of novel pathways oxygen is involved in, culminating with the award of the Nobel Prize for Medicine and Physiology in 2019 to the pioneers of this field, Greg Semenza, Peter Ratcliffe and William Kaelin. However, it is now beginning to be appreciated that oxygen can be a signalling molecule involved in a vast array of molecular processes, most of which impinge on gene expression control. This review will focus on the knowns and unknowns of oxygen as a signalling molecule, highlighting the role of 2-oxoglutarate-dependent dioxygenases as central players in the cellular response to deviations in oxygen tension.

HIF-1ß Positively Regulates NF-κB Activity via Direct Control of TRAF6.

NF-κB signalling is crucial for cellular responses to inflammation but is also associated with the hypoxia response. NF-κB and hypoxia inducible factor (HIF) transcription factors possess an intense molecular crosstalk. Although it is known that HIF-1α modulates NF-κB transcriptional response, very little is understood regarding how HIF-1ß contributes to NF-κB signalling. Here, we demonstrate that HIF-1ß is required for full NF-κB activation in cells following canonical and non-canonical stimuli. We found that HIF-1ß specifically controls TRAF6 expression in human cells but also in Drosophila melanogaster. HIF-1ß binds to the TRAF6 gene and controls its expression independently of HIF-1α. Furthermore, exogenous TRAF6 expression is able to rescue all of the cellular phenotypes observed in the absence of HIF-1ß. These results indicate that HIF-1ß is an important regulator of NF-κB with consequences for homeostasis and human disease.

Nestin expression in primary and metastatic uveal melanoma - possible biomarker for high-risk uveal melanoma.

PURPOSE: Nestin, a member of the intermediate filament protein family, has been described as a putative cancer stem cell marker (CSC) in uveal melanoma and poor prognostic factor in a variety of tumours, including cutaneous melanoma. In this study, we examined the expression of nestin in primary (PUM) and metastatic uveal melanoma (MUM) samples, and correlated the findings with histological, clinical and survival data. METHODS: Nestin expression was assessed by immunohistochemistry in 141 PUM and 26 MUM samples; 11 PUM cases were matched with their corresponding metastases. The percentage of tumour cells expressing nestin was scored by three independent observers. Statistical analysis of all data was performed with SPSS. RESULTS: Nestin expression was identified in both the cytoplasm and membrane of UM cells. Increased expression of nestin in PUM samples was associated with known poor prognostic parameters, including epithelioid cell morphology (p < 0.001), closed loops (p = 0.001), higher mitotic count (p < 0.001), monosomy 3 (p = 0.007) and chromosome 8q gain (p < 0.001). Primary uveal melanoma (PUM) with nestin expression levels above a cut-off value of 10% [as determined by receiver operating characteristic (ROC) analysis] was associated with a significantly reduced survival time (Log-rank, p = 0.002). In MUM, a higher percentage of nestin-positive tumour cells combined with poor prognostic markers in the PUM led to a shorter survival time following the development of metastases. CONCLUSION: In conclusion, increased nestin expression in PUM is a predictor of a tumour phenotype associated with metastatic progression and reduced survival time at onset of metastasis.