Hypoxia is a common denominator in the pathophysiology of a variety of human disease states. Insi... more Hypoxia is a common denominator in the pathophysiology of a variety of human disease states. Insight into how cells detect, and respond to low oxygen is crucial to understanding the role of hypoxia in disease. Central to the hypoxic response is rapid changes in the expression of genes essential to carry out a wide range of functions to adapt the cell/tissue to decreased oxygen availability. These changes in gene expression are co-ordinated by specialised transcription factors, changes to chromatin architecture and intricate balances between protein synthesis and destruction that together establish changes to the cellular proteome. In this article, we will discuss the advances of our understanding of the cellular oxygen sensing machinery achieved through the application of ‘omics-based experimental approaches.
Hypoxia—reduction in oxygen availability—plays key roles in both physiological and pathological p... more Hypoxia—reduction in oxygen availability—plays key roles in both physiological and pathological processes. Given the importance of oxygen for cell and organism viability, mechanisms to sense and respond to hypoxia are in place. A variety of enzymes utilise molecular oxygen, but of particular importance to oxygen sensing are the 2-oxoglutarate (2-OG) dependent dioxygenases (2-OGDs). Of these, Prolyl-hydroxylases have long been recognised to control the levels and function of Hypoxia Inducible Factor (HIF), a master transcriptional regulator in hypoxia, via their hydroxylase activity. However, recent studies are revealing that dioxygenases are involved in almost all aspects of gene regulation, including chromatin organisation, transcription and translation. We highlight the relevance of HIF and 2-OGDs in the control of gene expression in response to hypoxia and their relevance to human biology and health.
Reduced oxygen availability (hypoxia) can act as a signalling cue in physiological processes such... more 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) coordinate a transcriptional programme required for survival and adaptation to hypoxia. The effects of hypoxia and HIF on the chromatin accessibility landscape are still unclear. Here, using genome wide mapping of chromatin accessibility via ATAC-seq, we find hypoxia induces loci specific changes in chromatin accessibility enriched at hypoxia transcriptionally responsive genes. These changes are predominantly HIF dependent, reversible upon reoxygenation and partially mimicked by chemical HIF stabilisation independent of molecular dioxygenase inhibition. This work demonstrates that indeed, HIF stabilisation is necessary and sufficient to...
PBRM1, a component of the chromatin remodeller SWI/SNF, is often deleted or mutated in human canc... more PBRM1, a component of the chromatin remodeller SWI/SNF, is often deleted or mutated in human cancers, most prominently in renal cancers. Core components of the SWI/SNF complex have been shown to be important for the cellular response to hypoxia. Here we investigated how PBRM1 controls HIF-1alpha activity. We find that PBRM1 is required for HIF-1alpha transcriptional activity and protein levels. Mechanistically, PBRM1 is important for HIF-1alpha mRNA translation, as absence of PBRM1 results in reduced activly transalting HIF-1alpha mRNA. Interestingly, we find that PBRM1, but not BRG1, interacts with the m6A reader protein YTHDF2. HIF-1alpha mRNA is m6A modified, bound by PBRM1 and YTHDF2. PBRM1 is necessary for YTHDF2 binding to HIF-1alpha mRNA and reduction of YTHDF2 results in reduced HIF-1alpha protein expression in cells. Our results identify a SWI/SNF independent function for PBRM1, interacting with HIF-1alpha mRNA and the epitranscriptome machinery. Furthermore, our results su...
NF-κB signalling is crucial for cellular responses to inflammation but is also associated with th... more 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-1β contributes to NF-κB signalling. Here, we demonstrate that HIF-1β is required for full NF-κB activation in cells following canonical and non-canonical stimuli. We found that HIF-1β specifically controls TRAF6 expression in human cells but also in Drosophila melanogaster. HIF-1β 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-1β. These results indicate that HIF-1β is an important regulator of NF-κB with consequences for homeostasis and human disease.
NF-B signalling is crucial for cellular responses to inflammation but has also been associated w... more NF-B signalling is crucial for cellular responses to inflammation but has also been associated with the hypoxia response. NF-B and HIF transcription factors possess an intense molecular crosstalk. Although it is known that HIF-1beta modulates NF-kappaB transcriptional response, very little is understood regarding how HIF-1beta contributes to NF-kappaB signalling. Here, we demonstrate that HIF-1beta is required for full NF-kappaB activation in cells following canonical and non-canonical stimuli. We found that HIF-1beta specifically controls TRAF6 expression in human cells but also in Drosophila melanogaster. HIF-1beta binds to the TRAF6 gene and controls its expression independently of HIF-1alpha. Furthermore, exogenous TRAF6 expression is able to rescue all of the cellular phenotypes observed in the absence of HIF-1beta. These results indicate that HIF-1beta is an important regulator of NF-kappaB with consequences for homeostasis and human disease.
HIF (hypoxia-inducible factor) is the main transcription factor activated by low oxygen tensions.... more HIF (hypoxia-inducible factor) is the main transcription factor activated by low oxygen tensions. HIF-1α (and other α subunits) is tightly controlled mostly at the protein level, through the concerted action of a class of enzymes called PHDs (prolyl hydroxylases) 1, 2 and 3. Most of the knowledge of HIF derives from studies following hypoxic stress; however, HIF-1α stabilization is also found in non-hypoxic conditions through an unknown mechanism. In the present study, we demonstrate that NF-κB (nuclear factor κB) is a direct modulator of HIF-1α expression. The HIF-1α promoter is responsive to selective NF-κB subunits. siRNA (small interfering RNA) studies for individual NF-κB members revealed differential effects on HIF-1α mRNA levels, indicating that NF-κB can regulate basal HIF-1α expression. Finally, when endogenous NF-κB is induced by TNFα (tumour necrosis factor α) treatment, HIF-1α levels also change in an NF-κB-dependent manner. In conclusion, we find that NF-κB can regulate...
The cell cycle is an important cellular process whereby the cell attempts to replicate its genome... more 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.
Hypoxia — reduction in oxygen availability—plays key roles in both physiological and pathological... more Hypoxia — reduction in oxygen availability—plays key roles in both physiological and pathological processes. Given the importance of oxygen for cell and organism viability, mechanisms to sense and respond to hypoxia are in place. A variety of enzymes utilise molecular oxygen, but of particular importance to oxygen sensing are the 2-oxoglutarate (2-OG) dependent dioxygenases (2OGDs). Of these, Prolyl-hydroxylases have long been recognised to control the levels and function of Hypoxia Inducible Factor (HIF), a master transcriptional regulator in hypoxia, via their hydroxylase activity. However, recent studies are revealing that dioxygenases are involved in almost all aspects of gene regulation, including chromatin organisation, transcription and translation. We highlight the relevance of HIF and 2-OG dioxyenases in the control of gene expression in response to hypoxia and their relevance to human cancers.
PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in... more PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression through the regulation of the crucial centrosomal protein Cep192, establishing a link between the oxygen-sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, CDK4 and CDK6 at S130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of hypoxia-inducible factor (HIF) protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, although phosphorylation of PHD1 at S130 lowers its activity towards HIF1α, this modification increases the activity of PHD1 towards Cep192. These results establish a mechanism by which ...
The SIN3A-HDAC complex is a master transcriptional repressor, required for development but often ... more The SIN3A-HDAC complex is a master transcriptional repressor, required for development but often deregulated in disease. Here, we report that the recently identified new component of this complex, SINHCAF/FAM60A, links the SIN3A-HDAC co-repressor complex function to the hypoxia response. SINHCAF Chromatin Immunoprecipitation-sequencing and gene expression analysis reveal a signature associated with the activation of the hypoxia response. We show that SINHCAF specifically repress HIF 2α mRNA and protein expression resulting in functional cellular changes in in-vitro angiogenesis, and proliferation. Analysis of patient datasets demonstrates that SINHCAF and HIF 2α mRNA levels are inversely correlated and predict contrasting outcomes for patient survival in both colon and lung cancer. This relationship is also observed in a mouse model of colon cancer, indicating an evolutionary conserved mechanism. Our analysis reveals an unexpected link between SINHCAF and cancer cell signalling via ...
Hypoxia is not only a developmental cue but also a stress and pathological stimulus in many human... more Hypoxia is not only a developmental cue but also a stress and pathological stimulus in many human diseases. The response to hypoxia at the cellular level relies on the activity of the transcription factor family, hypoxia inducible factor (HIF). HIF-1 is responsible for the acute response and transactivates a variety of genes involved in cellular metabolism, cell death, and cell growth. Here, we show that hypoxia results in increased mRNA levels for human lysine (K)-specific demethylase 2 (KDM2) family members, KDM2A and KDM2B, and also for Drosophila melanogaster KDM2, a histone and protein demethylase. In human cells, KDM2 family member's mRNA levels are regulated by HIF-1 but not HIF-2 in hypoxia. Interestingly, only KDM2A protein levels are significantly induced in a HIF-1-dependent manner, while KDM2B protein changes in a cell type-dependent manner. Importantly, we demonstrate that in human cells, KDM2A regulation by hypoxia and HIF-1 occurs at the level of promoter, with HI...
Cancer is often characterised by the presence of hypoxia and inflammation. Paramount to the mecha... more Cancer is often characterised by the presence of hypoxia and inflammation. Paramount to the mechanisms controlling cellular responses under such stress stimuli, are the transcription factor families of Hypoxia Inducible Factor (HIF) and Nuclear Factor of κ-light-chain-enhancer of activated B cells (NF-κB). Although, a detailed understating of how these transcription factors respond to their cognate stimulus is well established, it is now appreciated that HIF and NF-κB undergo extensive crosstalk, in particular in pathological situations such as cancer. Here, we focus on the current knowledge on how HIF is activated by inflammation and how NF-κB is modulated by hypoxia. We summarise the evidence for the possible mechanism behind this activation and how HIF and NF-κB function impacts cancer, focusing on colorectal, breast and lung cancer. We discuss possible new points of therapeutic intervention aiming to harness the current understanding of the HIF-NF-κB crosstalk.
PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in... more PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression through the regulation of the crucial centrosomal protein Cep192, establishing a link between the oxygen-sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, CDK4 and CDK6 at S130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of hypoxia-inducible factor (HIF) protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, although phosphorylation of PHD1 at S130 lowers its activity towards HIF1α, this modification increases the activity of PHD1 towards Cep192. These results establish a mechanism by which ...
Mechanisms regulating protein degradation ensure the correct and timely expression of transcripti... more Mechanisms regulating protein degradation ensure the correct and timely expression of transcription factors such as hypoxia inducible factor (HIF). Under normal oxygen tensions, HIFα subunits are targeted for proteasomal degradation mainly by vHL-dependent ubiquitination. Deubiquitinases are responsible for reversing this process. While the mechanism and regulation of HIFα ubiquitin-dependent proteasomal degradation has been the object of many studies, little is known about the role of deubiquitinases. Here we show that HIF2α expression is regulated by the deubiquitinase Cezanne in an E2F1-dependent manner. Knockdown of Cezanne down-regulates HIF2α mRNA, protein and activity independently of hypoxia and proteasomal degradation. Mechanistically, HIF2α gene expression is controlled directly by E2F1, and Cezanne regulates the stability of E2F1. Exogenous E2F1 can rescue HIF2α transcript and protein expression, when Cezanne is depleted. Together these data reveal a novel mechanism for t...
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since t... more In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
Hypoxia is a common denominator in the pathophysiology of a variety of human disease states. Insi... more Hypoxia is a common denominator in the pathophysiology of a variety of human disease states. Insight into how cells detect, and respond to low oxygen is crucial to understanding the role of hypoxia in disease. Central to the hypoxic response is rapid changes in the expression of genes essential to carry out a wide range of functions to adapt the cell/tissue to decreased oxygen availability. These changes in gene expression are co-ordinated by specialised transcription factors, changes to chromatin architecture and intricate balances between protein synthesis and destruction that together establish changes to the cellular proteome. In this article, we will discuss the advances of our understanding of the cellular oxygen sensing machinery achieved through the application of ‘omics-based experimental approaches.
Hypoxia—reduction in oxygen availability—plays key roles in both physiological and pathological p... more Hypoxia—reduction in oxygen availability—plays key roles in both physiological and pathological processes. Given the importance of oxygen for cell and organism viability, mechanisms to sense and respond to hypoxia are in place. A variety of enzymes utilise molecular oxygen, but of particular importance to oxygen sensing are the 2-oxoglutarate (2-OG) dependent dioxygenases (2-OGDs). Of these, Prolyl-hydroxylases have long been recognised to control the levels and function of Hypoxia Inducible Factor (HIF), a master transcriptional regulator in hypoxia, via their hydroxylase activity. However, recent studies are revealing that dioxygenases are involved in almost all aspects of gene regulation, including chromatin organisation, transcription and translation. We highlight the relevance of HIF and 2-OGDs in the control of gene expression in response to hypoxia and their relevance to human biology and health.
Reduced oxygen availability (hypoxia) can act as a signalling cue in physiological processes such... more 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) coordinate a transcriptional programme required for survival and adaptation to hypoxia. The effects of hypoxia and HIF on the chromatin accessibility landscape are still unclear. Here, using genome wide mapping of chromatin accessibility via ATAC-seq, we find hypoxia induces loci specific changes in chromatin accessibility enriched at hypoxia transcriptionally responsive genes. These changes are predominantly HIF dependent, reversible upon reoxygenation and partially mimicked by chemical HIF stabilisation independent of molecular dioxygenase inhibition. This work demonstrates that indeed, HIF stabilisation is necessary and sufficient to...
PBRM1, a component of the chromatin remodeller SWI/SNF, is often deleted or mutated in human canc... more PBRM1, a component of the chromatin remodeller SWI/SNF, is often deleted or mutated in human cancers, most prominently in renal cancers. Core components of the SWI/SNF complex have been shown to be important for the cellular response to hypoxia. Here we investigated how PBRM1 controls HIF-1alpha activity. We find that PBRM1 is required for HIF-1alpha transcriptional activity and protein levels. Mechanistically, PBRM1 is important for HIF-1alpha mRNA translation, as absence of PBRM1 results in reduced activly transalting HIF-1alpha mRNA. Interestingly, we find that PBRM1, but not BRG1, interacts with the m6A reader protein YTHDF2. HIF-1alpha mRNA is m6A modified, bound by PBRM1 and YTHDF2. PBRM1 is necessary for YTHDF2 binding to HIF-1alpha mRNA and reduction of YTHDF2 results in reduced HIF-1alpha protein expression in cells. Our results identify a SWI/SNF independent function for PBRM1, interacting with HIF-1alpha mRNA and the epitranscriptome machinery. Furthermore, our results su...
NF-κB signalling is crucial for cellular responses to inflammation but is also associated with th... more 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-1β contributes to NF-κB signalling. Here, we demonstrate that HIF-1β is required for full NF-κB activation in cells following canonical and non-canonical stimuli. We found that HIF-1β specifically controls TRAF6 expression in human cells but also in Drosophila melanogaster. HIF-1β 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-1β. These results indicate that HIF-1β is an important regulator of NF-κB with consequences for homeostasis and human disease.
NF-B signalling is crucial for cellular responses to inflammation but has also been associated w... more NF-B signalling is crucial for cellular responses to inflammation but has also been associated with the hypoxia response. NF-B and HIF transcription factors possess an intense molecular crosstalk. Although it is known that HIF-1beta modulates NF-kappaB transcriptional response, very little is understood regarding how HIF-1beta contributes to NF-kappaB signalling. Here, we demonstrate that HIF-1beta is required for full NF-kappaB activation in cells following canonical and non-canonical stimuli. We found that HIF-1beta specifically controls TRAF6 expression in human cells but also in Drosophila melanogaster. HIF-1beta binds to the TRAF6 gene and controls its expression independently of HIF-1alpha. Furthermore, exogenous TRAF6 expression is able to rescue all of the cellular phenotypes observed in the absence of HIF-1beta. These results indicate that HIF-1beta is an important regulator of NF-kappaB with consequences for homeostasis and human disease.
HIF (hypoxia-inducible factor) is the main transcription factor activated by low oxygen tensions.... more HIF (hypoxia-inducible factor) is the main transcription factor activated by low oxygen tensions. HIF-1α (and other α subunits) is tightly controlled mostly at the protein level, through the concerted action of a class of enzymes called PHDs (prolyl hydroxylases) 1, 2 and 3. Most of the knowledge of HIF derives from studies following hypoxic stress; however, HIF-1α stabilization is also found in non-hypoxic conditions through an unknown mechanism. In the present study, we demonstrate that NF-κB (nuclear factor κB) is a direct modulator of HIF-1α expression. The HIF-1α promoter is responsive to selective NF-κB subunits. siRNA (small interfering RNA) studies for individual NF-κB members revealed differential effects on HIF-1α mRNA levels, indicating that NF-κB can regulate basal HIF-1α expression. Finally, when endogenous NF-κB is induced by TNFα (tumour necrosis factor α) treatment, HIF-1α levels also change in an NF-κB-dependent manner. In conclusion, we find that NF-κB can regulate...
The cell cycle is an important cellular process whereby the cell attempts to replicate its genome... more 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.
Hypoxia — reduction in oxygen availability—plays key roles in both physiological and pathological... more Hypoxia — reduction in oxygen availability—plays key roles in both physiological and pathological processes. Given the importance of oxygen for cell and organism viability, mechanisms to sense and respond to hypoxia are in place. A variety of enzymes utilise molecular oxygen, but of particular importance to oxygen sensing are the 2-oxoglutarate (2-OG) dependent dioxygenases (2OGDs). Of these, Prolyl-hydroxylases have long been recognised to control the levels and function of Hypoxia Inducible Factor (HIF), a master transcriptional regulator in hypoxia, via their hydroxylase activity. However, recent studies are revealing that dioxygenases are involved in almost all aspects of gene regulation, including chromatin organisation, transcription and translation. We highlight the relevance of HIF and 2-OG dioxyenases in the control of gene expression in response to hypoxia and their relevance to human cancers.
PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in... more PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression through the regulation of the crucial centrosomal protein Cep192, establishing a link between the oxygen-sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, CDK4 and CDK6 at S130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of hypoxia-inducible factor (HIF) protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, although phosphorylation of PHD1 at S130 lowers its activity towards HIF1α, this modification increases the activity of PHD1 towards Cep192. These results establish a mechanism by which ...
The SIN3A-HDAC complex is a master transcriptional repressor, required for development but often ... more The SIN3A-HDAC complex is a master transcriptional repressor, required for development but often deregulated in disease. Here, we report that the recently identified new component of this complex, SINHCAF/FAM60A, links the SIN3A-HDAC co-repressor complex function to the hypoxia response. SINHCAF Chromatin Immunoprecipitation-sequencing and gene expression analysis reveal a signature associated with the activation of the hypoxia response. We show that SINHCAF specifically repress HIF 2α mRNA and protein expression resulting in functional cellular changes in in-vitro angiogenesis, and proliferation. Analysis of patient datasets demonstrates that SINHCAF and HIF 2α mRNA levels are inversely correlated and predict contrasting outcomes for patient survival in both colon and lung cancer. This relationship is also observed in a mouse model of colon cancer, indicating an evolutionary conserved mechanism. Our analysis reveals an unexpected link between SINHCAF and cancer cell signalling via ...
Hypoxia is not only a developmental cue but also a stress and pathological stimulus in many human... more Hypoxia is not only a developmental cue but also a stress and pathological stimulus in many human diseases. The response to hypoxia at the cellular level relies on the activity of the transcription factor family, hypoxia inducible factor (HIF). HIF-1 is responsible for the acute response and transactivates a variety of genes involved in cellular metabolism, cell death, and cell growth. Here, we show that hypoxia results in increased mRNA levels for human lysine (K)-specific demethylase 2 (KDM2) family members, KDM2A and KDM2B, and also for Drosophila melanogaster KDM2, a histone and protein demethylase. In human cells, KDM2 family member's mRNA levels are regulated by HIF-1 but not HIF-2 in hypoxia. Interestingly, only KDM2A protein levels are significantly induced in a HIF-1-dependent manner, while KDM2B protein changes in a cell type-dependent manner. Importantly, we demonstrate that in human cells, KDM2A regulation by hypoxia and HIF-1 occurs at the level of promoter, with HI...
Cancer is often characterised by the presence of hypoxia and inflammation. Paramount to the mecha... more Cancer is often characterised by the presence of hypoxia and inflammation. Paramount to the mechanisms controlling cellular responses under such stress stimuli, are the transcription factor families of Hypoxia Inducible Factor (HIF) and Nuclear Factor of κ-light-chain-enhancer of activated B cells (NF-κB). Although, a detailed understating of how these transcription factors respond to their cognate stimulus is well established, it is now appreciated that HIF and NF-κB undergo extensive crosstalk, in particular in pathological situations such as cancer. Here, we focus on the current knowledge on how HIF is activated by inflammation and how NF-κB is modulated by hypoxia. We summarise the evidence for the possible mechanism behind this activation and how HIF and NF-κB function impacts cancer, focusing on colorectal, breast and lung cancer. We discuss possible new points of therapeutic intervention aiming to harness the current understanding of the HIF-NF-κB crosstalk.
PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in... more PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression through the regulation of the crucial centrosomal protein Cep192, establishing a link between the oxygen-sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, CDK4 and CDK6 at S130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of hypoxia-inducible factor (HIF) protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, although phosphorylation of PHD1 at S130 lowers its activity towards HIF1α, this modification increases the activity of PHD1 towards Cep192. These results establish a mechanism by which ...
Mechanisms regulating protein degradation ensure the correct and timely expression of transcripti... more Mechanisms regulating protein degradation ensure the correct and timely expression of transcription factors such as hypoxia inducible factor (HIF). Under normal oxygen tensions, HIFα subunits are targeted for proteasomal degradation mainly by vHL-dependent ubiquitination. Deubiquitinases are responsible for reversing this process. While the mechanism and regulation of HIFα ubiquitin-dependent proteasomal degradation has been the object of many studies, little is known about the role of deubiquitinases. Here we show that HIF2α expression is regulated by the deubiquitinase Cezanne in an E2F1-dependent manner. Knockdown of Cezanne down-regulates HIF2α mRNA, protein and activity independently of hypoxia and proteasomal degradation. Mechanistically, HIF2α gene expression is controlled directly by E2F1, and Cezanne regulates the stability of E2F1. Exogenous E2F1 can rescue HIF2α transcript and protein expression, when Cezanne is depleted. Together these data reveal a novel mechanism for t...
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since t... more In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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Papers by Sonia Rocha