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Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN
Contributor:Dąbrowski, Michał (1969– ) : Supervisor ; Charzyńska, Agata : Auxiliary supervisor
Publisher:Nencki Institute of Experimental Biology PAS
Place of publishing: Date issued/created: Description:116 pages : illustrations ; 30 cm ; Bibliography ; Summary in Polish
Degree name: Degree discipline : Degree grantor:Nencki Institute of Experimental Biology PAS ; degree obtained: 28/06/2024
Type of object: Subject and Keywords:Endothelium ; HIF1 ; HIF2 ; Hypoxia
Abstract:
Cellular response to hypoxia is regulated by hypoxia-inducible transcription factors called HIFs. Those transcription factors are heterodimers made of two HIF subunits: constitutively expressed beta subunit (HIF1B) and oxygen-dependent alpha subunits, of which there are three major isoforms: HIF1A encoded by HIF1A, HIF2A encoded by the EPAS1, and HIF3A encoded by HIF3A. HIF1A is responsible for the acute response to hypoxia, whereas HIF2A and HIF3A are responsible for the adaptation to the long-term hypoxia. During oxygen homeostasis, the concentration of the alpha subunits is low, due to their oxygen-dependent degradation. During hypoxia, this degradation process is interrupted, which leads to the accumulation of alpha subunits, their translocation to the nucleus, where they dimerize with HIF1B to form transcriptionally active complexes. Active HIF complexes bind to hypoxia-response elements (HREs) in target-gene promoters to regulate their response to hypoxia. HIF1 and HIF2 regulate the adaptation of vascular endothelial cells to low oxygen conditions, by activating signalling pathways and genes, which are responsible for endothelial cells migration, growth, differentiation and metabolism. In this dissertation, I characterised two previously described HRE motifs annotated to HIF1 and HIF2, by identifying their instances in the open chromatin regions in promoters of hypoxia-resposive genes, their association with the timepoint of gene activation under hypoxia, and their spatial distribution in the promoters of hypoxia-responsive genes. These results confirmed that the two HRE motifs do have some specificity for HIF1 and HIF2. We investigated the effects of silencing of either HIF1A or HIF2A in Human Umbilical Vein Endothelial Cells (HUVECs) on the expression of 14 pre-selected hypoxia-responsive genes. Among these genes, we identified genes that in HUVECs are regulated by HIF1 (ANKRD37, NARF, BNIP3, SLC2A1), by HIF2 (ADM, ANGPTL4, C1orf21, MAGI1, PTGIS), and by both HIF1 and HIF2 (EGLN3, LUCAT1, MIR210HG, BNIP3L), in the time-window when both HIF1 and HIF2 are active. I demonstrated a linear proportionality between the effect of HIF1 on gene activation and the count of HRE motifs annotated to HIF1 in promoter open chromatin regions. I corroborated this result by genome-wide analysis of HRE motif content in normoxic HUVECs open chromatin regions and HIF1A binding in these cells under hypoxia. This allowed us to propose a mechanism, by which higher content of HRE motifs annotated to HIF1 in open chromatin regions increases HIF1 binding, which contributes to increased gene induction due to HIF1 under hypoxia. I also report that for 232 previously identified hypoxia-responsive genes, the genes which have in their promoter regions ChIP-seq peaks for HIF1A contain more HRE motifs annotated to HIF1A, than genes which do not contain said ChIP-seq peaks in their promoter regions. I developed an ordinary differential equations (ODE) model of hypoxia signalling and transcriptional activation of hypoxia responsive genes that takes into account not only HIF1 but also HIF2. Within this model, I was able to correctly simulate the effects of a further drop of oxygen level during hypoxia on the HIF switch. These simulations results support experimentally established conclusion that residual PHD activity under hypoxia contributes to the HIF-switch. Furthermore, by simulations in the model I established that, for the simulation results to broadly agree with experiments, there is a need for a large excess of HIF1B over the two HIF alpha subunits. However, our model including both HIFs was not better than model including only HIF1 in predicting mRNA expression of hypoxia responsive genes. The results described in this dissertation illustrate the relationship between the type and number of HRE motifs in open chromatin regions in the promoters of hypoxia responsive genes and their transcriptional activation by HIF1 and HIF2.
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Digitizing institution:Nencki Institute of Experimental Biology of the Polish Academy of Sciences
Original in:Library of the Nencki Institute of Experimental Biology PAS
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