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HIF1A (Hypoxia-inducible factor one alpha)

Mechanism summary

Figure 1: Signaling pathway of HIF1A.

Hypoxia-inducible factors (HIFs) are transcription factors responding to decreased oxygen levels in the cellular environment. Hypoxia-inducible factor 1-alpha (HIF1A) is constantly expressed in mammalian cells. Under normoxic conditions HIF1A is hydroxylated and is marked by the E3 ubiquitin ligase which leads to the degradation by the proteasome. In hypoxic conditions HIF1A is stabilized and can heterodimerize with HIF1B. Also HIF1A transcription is often significantly upregulated under hypoxic conditions. Under hypoxic conditions HIF1 can then bind to hypoxia response elements (HREs) (BBa_K2295003) in the nucleus (Ziello et al., 2007).

Structural and Functional Overview

• added by team Latvia-Riga 2024 (https://teams.igem.org/5415)

Introduction

Hypoxia–inducible factor 1–alpha (HIF–1α) is a crucial transcription factor that plays a vital role in cellular responses to low oxygen levels (hypoxia). Structurally, HIF–1α is a basic helix–loop–helix (bHLH) protein characterised by its dimerization with HIF–1β (also known as ARNT) to form a heterodimeric complex [1][2][3]. This complex is essential for binding to hypoxia response elements (HREs) in the promoters of target genes, which include those involved in angiogenesis, erythropoiesis, and metabolic adaptation [1][2].

The HIF–1α protein comprises several key structural domains: an N–terminal oxygen–dependent degradation domain (ODD), a bHLH domain, a PAS–A and PAS–B (Per–Arnt–Sim) heterodimerization and specificity enhancing domains, and a C–terminal transactivation domain [3][4]. The ODD is particularly important as it contains prolyl residues that are hydroxylated under normoxic conditions, leading to the binding of the von Hippel–Lindau (VHL) protein and subsequent proteasomal degradation of HIF–1α [1][4]. Such regulation ensures that HIF–1α levels are tightly controlled in response to oxygen availability.

Under hypoxic conditions, the hydroxylation of specific proline residues (Pro402 and Pro564) is inhibited, allowing HIF–1α to accumulate and translocate to the nucleus [1][2][3]. Once in the nucleus, HIF–1α dimerizes with HIF–1β, and the complex binds to HREs, initiating the transcription of over 60 genes, critical for adaptation to low oxygen levels [1][2][3]. The basic domain of HIF–1α facilitates DNA binding, while the transactivation domain recruits co–activators that enhance transcriptional activity [3][4]. Additionally, HIF–1α is subject to various post–translational modifications, including acetylation and phosphorylation, which further modulate its activity and stability [5][6].

HIF–1alpha protein target genes

Research has identified a multitude of target genes regulated by HIF–1α, underscoring its profound impact on cellular physiology. These target genes not only facilitate the adaptive response to hypoxia but also play critical roles in various pathological conditions. The dysregulation of HIF–1α and its associated target genes is frequently linked to tumour progression, metastasis, and resistance to therapeutic interventions [10]. Listed below are some of the most prominent HIF–1alpha protein target genes and their functional significance in cancer pathogenesis.

Vascular Endothelial Growth Factor (VEGF)

Primary regulatory gene of angiogenesis, promoting the formation of new blood vessels to supply oxygen and nutrients to tumours. Increased VEGF expression is often linked to poor clinical outcomes in cancer patients due to enhanced tumour vascularization and metastasis [1][7][8].

Glucose Transporter genes (GLUTs)

HIF–1α upregulates glucose transporters, particularly GLUT1 and GLUT3, which facilitate increased glucose uptake in hypoxic cells. This adaptation is vital for supporting the Warburg effect, where cancer cells rely on glycolysis for energy production even in the presence of oxygen [1][8].

Glycolytic Enzymes (GEs)

HIF–1α enhances the expression of key glycolytic enzymes, including lactate dehydrogenase A (LDH–A), phosphofructokinase, and hexokinase, which facilitate anaerobic metabolism under low oxygen conditions. This metabolic shift is crucial for tumour cell survival and proliferation in hypoxic microenvironments [6][8].

Erythropoietin (EPO)

A hormone that stimulates red blood cell production, enhancing oxygen delivery. HIF–1α–mediated EPO expression is vital for maintaining systemic oxygen homeostasis, particularly in conditions of chronic hypoxia [1][6].

Inducible Nitric Oxide Synthase (iNOS)

Enzyme involved in the production of nitric oxide, which plays a role in angiogenesis and immune response. HIF–1α upregulates iNOS, contributing to the tumour microenvironment's angiogenic and immunosuppressive properties [1][8].

Heme Oxygenase–1 (HO–1)

HO–1 plays a protective role against oxidative stress, which is often exacerbated in hypoxic conditions. HIF–1α upregulates HO–1 expression, contributing to cell survival and adaptation under adverse conditions. The protective effects of HO–1 are particularly relevant in cancer, where oxidative stress can promote tumour progression [8][9][10].

HIF–1 alpha coactivator and corepressor interactions

The transcriptional activity of HIF–1α is finely tuned by its interactions with various co–activators and co–repressors, the balance between which is critical in modulating its ability to initiate and sustain proper hypoxia responsive gene expression.

One of the most prominent co–activators associated with HIF–1α is p300/CBP (CREB–binding protein), which possesses histone acetyltransferase activity. Upon stabilisation of HIF–1α under hypoxic conditions, p300/CBP is recruited to the HIF–1 complex, leading to increased acetylation of histones at HIF target gene promoters. This modification promotes a more relaxed chromatin structure, making it accessible for RNA polymerase II and other transcription factors necessary for transcription initiation. Additionally, other co–activators such as the Super Elongation Complex (SEC), which includes components like CDK8 and AFF4, are also involved in enhancing transcriptional elongation at HIF target genes [13][9][8].

Long non–coding RNAs (lncRNAs) have also been identified as important co–activators of HIF–1α. For instance, LncHIFCAR has been shown to facilitate the interaction between HIF–1α and its co–activators, enhancing the assembly of the HIF–1 transcriptional complex. This lncRNA not only stabilises HIF–1α but also promotes its binding to hypoxia response elements (HREs) in target gene promoters, thereby amplifying the transcriptional response to hypoxia [12][13].

Conversely, proteins such as COMMD1 and EAF2 serve as corepressors that can disrupt the heterodimerization between HIF–1α and HIF–1β, thereby preventing effective binding to DNA. Other corepressors may recruit histone deacetylases (HDACs) or other chromatin–modifying enzymes that lead to a closed chromatin conformation, further inhibiting transcription [9][14].

Post–translational modulation of HIF–1alpha

The most well–studied post–translational modulatory processes (PTMs) of HIF–1α are hydroxylation, acetylation, and phosphorylation. Under normoxic conditions, HIF–1α is hydroxylated on specific proline residues (Pro402 and Pro564) within its oxygen–dependent degradation (ODD) domain by prolyl hydroxylase domain (PHD) enzymes [11][12]. This hydroxylation allows the von Hippel–Lindau (VHL) protein to bind to HIF–1α, leading to its polyubiquitination and subsequent proteasomal degradation [9][11]. Acetylation of Lys532 within the ODD domain by the acetyltransferase ARD1 also promotes VHL binding and HIF–1α degradation under normoxia [11].

In contrast, under hypoxic conditions, PHD enzymes are inhibited, preventing HIF–1α hydroxylation and allowing it to accumulate in the nucleus [11]. Once stabilised, HIF–1α can dimerize with HIF–1β and bind to hypoxia response elements (HREs) in the promoters of target genes [11][12]. Acetylation of HIF–1α by the p300/CBP coactivators enhances its transcriptional activity by promoting the recruitment of the transcriptional machinery [9][11]. Phosphorylation of HIF–1α by various kinases, such as MAPKs and GSK3β, can also modulate its stability and activity [11]. Phosphorylation can either enhance or inhibit HIF–1α transcriptional activity, depending on the specific residues modified and the cellular context [11].

Among the less commonly recognized post–translational modifications (PTMs) of HIF–1α are sumoylation and S–nitrosylation. SUMOylation on Lys391 and Lys477 in the ODD domain may increase HIF–1α stability by competing with hydroxylation and acetylation for VHL binding [9]. S–nitrosylation of HIF–1α can enhance its transcriptional activity by promoting its interaction with p300/CBP [11].

References:

[1] Xiao, H., Gu, Z., Wang, G., Zhao, T. (2013). The Possible Mechanisms Underlying the Impairment of HIF-1α Pathway Signalling in Hyperglycemia and the Beneficial Effects of Certain Therapies. International Journal of Medical Sciences, 10(10), 1412-1421. https://doi.org/10.7150/ijms.5630.

[2] Lisy, K., Peet, D. (2008). Turn me on: regulating HIF transcriptional activity. Cell Death Differ 15, 642–649. https://doi.org/10.1038/sj.cdd.4402315.

[3] Masoud, G. N., & Li, W. (2015). HIF-1α pathway: role, regulation and intervention for cancer therapy. Acta pharmaceutica Sinica. B, 5(5), 378–389. https://doi.org/10.1016/j.apsb.2015.05.007

[4] Hu, C. J., Wang, L. Y., Chodosh, L. A., Keith, B., & Simon, M. C. (2003). Differential roles of hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hypoxic gene regulation. Molecular and cellular biology, 23(24), 9361–9374. https://doi.org/10.1128/MCB.23.24.9361-9374.2003

[5] Ziello, J.E., Jovin, I.S. and Huang, Y. (2007) Hypoxia-inducible factor (HIF)-1 regulatory pathway and its potential for therapeutic intervention in malignancy and ischemia, The Yale journal of biology and medicine. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2140184/.

[6] Masoud, G. N., Li, W. (2015). HIF-1α pathway: role, regulation and intervention for cancer therapy, Acta Pharmaceutica Sinica B, Volume 5, Issue 5, Pages 378-389, ISSN 2211-3835, https://doi.org/10.1016/j.apsb.2015.05.007.

[7] Ortmann, B. M. (2024) Hypoxia-inducible factor in cancer: from pathway regulation to therapeutic opportunity. BMJ Oncology 2024;3:e000154. https://bmjoncology.bmj.com/content/3/1/e000154.

[8] Hong, S. S., Lee, H., & Kim, K. W. (2004). HIF-1alpha: a valid therapeutic target for tumour therapy. Cancer research and treatment, 36(6), 343–353. https://doi.org/10.4143/crt.2004.36.6.343.

[9] Dengler, V. L., Galbraith, M., & Espinosa, J. M. (2014). Transcriptional regulation by hypoxia inducible factors. Critical reviews in biochemistry and molecular biology, 49(1), 1–15. https://doi.org/10.3109/10409238.2013.838205.

[10] Benita, Y., Kikuchi, H., Smith, A. D., Zhang, M. Q., Chung, D. C., & Xavier, R. J. (2009). An integrative genomics approach identifies Hypoxia Inducible Factor-1 (HIF-1)-target genes that form the core response to hypoxia. Nucleic acids research, 37(14), 4587–4602. https://doi.org/10.1093/nar/gkp425.

[11] Albanese, A., Daly, L. A., Mennerich, D., Kietzmann, T., & Sée, V. (2020). The Role of Hypoxia-Inducible Factor Post-Translational Modifications in Regulating Its Localisation, Stability, and Activity. International journal of molecular sciences, 22(1), 268. https://doi.org/10.3390/ijms22010268.

[12] Zheng, F., Chen, J., Zhang, X. et al. (2021). The HIF-1α antisense long non-coding RNA drives a positive feedback loop of HIF-1α mediated transactivation and glycolysis. Nat Commun 12, 1341. https://doi.org/10.1038/s41467-021-21535-3.

[13] Yfantis, A., Mylonis, I., Chachami, G., Nikolaidis, M., Amoutzias, G. D., Paraskeva, E., & Simos, G. (2023). Transcriptional Response to Hypoxia: The Role of HIF-1-Associated Co-Regulators. Cells, 12(5), 798. https://doi.org/10.3390/cells12050798.

[14] Lee, JW., Bae, SH., Jeong, JW. et al. (2004). Hypoxia-inducible factor (HIF-1)α: its protein stability and biological functions. Exp Mol Med 36, 1–12. https://doi.org/10.1038/emm.2004.1

Western Blot against HIF1A

Figure 2: Transient introduction of artificial HIF1A in HIF1A shRNA2 knockdown HEK293T cell line.

Induction of the overexpressed HIF1A-HA can be observed in the last lane. Endogenous and artificial HIF1A were detected with HIF1A antibody; reintroduced HIF1A was detected via an HA-tag; GAPDH served as a loading control. Wild type (WT) and knockdown (HIF1A shRNA2) cells were transfected with CMV:HIF1A-HA one day prior to induction with CoCl₂ to simulate hypoxia. Cells were harvested 24 hours after induction. Immunoblot assay was performed using antibodies against HIF1A, HA-tag and GAPDH.

Sequence and Features