Project 1: Nuclear actin dynamics in hypoxia- a role in transcription?

PI Göttingen: D.M. Katschinski, V.V. Belousov (Mercator Fellow); PI London: A.M. Shah

Scientific background and preliminary results

The hypoxia-inducible factors HIF-1 and HIF-2 are transcription factors are critically important for the transcriptional response to low oxygen availability (hypoxia). Hypoxia plays a central role in many physiological (e.g. development, growth, adaption to altitude) and pathophysiological processes (e.g. ischemic diseases, wound healing, inflammation, and cancer) (Semenza, 2012). Activation of the HIF-pathway is the classical way of cells to adapt to hypoxia. HIFs consist of a stable b-subunit that is located within the nucleus and a labile oxygen-regulated a-subunit. In normoxia, HIFa is constantly hydroxylated by PHDs (prolyl-hydroxylase domain enzymes) and FIH (factor inhibiting HIF) enzymes, ubiquitinated by pVHL and subsequently degraded. Under hypoxic conditions the PHD enzymes are inactive, HIFa is stabilized, shuttles into the nucleus where it dimerizes with its b- subunit. The HIF-dimer binds to hypoxia-responsive elements (HREs) which can be localized within the promoter region of genes, however can also be found far (several kb) away. Upon oxygen deprivation HIF-1a protein accumulates within 2 min in the nucleus (Jewell et al., 2001). However, in the overall adaptive transcriptional response to hypoxia, transcriptional repression is an equally important process. Still, underlying mechanisms are much less understood. Transcription can be repressed through several mechanisms: inhibition of the transcriptional machinery, inhibition of transcriptional activators or chromatin remodeling that changes the chromatin accessibility (Gaston and Jayaraman, 2003). In hypoxia gene repression leads to a reduction in energy demanding cellular processes.

Actin is a central component of the eukaryotic cytoskeleton and is involved in a wide variety of cellular processes including cell cytokinesis, membrane dynamics and cell motility. Besides having a well-established role as part of the cytoskeleton, actin is also present in the nucleus where it participates in fundamental nuclear processes. Nuclear actin is involved in histone-modifications and plays a part in chromatin remodeling complexes (Kapoor and Shen, 2014). It also has a role in transcriptional regulation by all three RNA polymerases (Visa and Percipalle, 2010) and binds to transcription factors or transcriptional co-activators and regulates their activity (Grosse and Vartiainen, 2013).

Hypotheses of the PhD project

We and others have observed changing actin dynamics in the cytoplasm in hypoxia (Leinhos et al., 2019, Zieseniss 2014), but up to now, hypoxic nuclear actin dynamics have not been investigated. We have observed the rapid depletion of the nuclear actin pool in hypoxia in our preliminary work and propose to follow this up with the goal to:

-              Analyze if hypoxic nuclear actin reduction influences gene expression.

We hypothesize that actin driven, HIF-independent non-canonical signaling events set the stage for fast global changes in the transcriptional program early in hypoxia.

Work programme


Visualization of actin organization and dynamics during hypoxia

Preliminary results suggest that the nuclear actin pool diminishes at the onset of hypoxia. To further investigate the temporal resolution and spatial dynamics of actin organization within hypoxic cells, the cells will be transfected with actin-directed nanobodies (Plessner et al., 2015). Fluorescent protein-tagged actin nanobodies recognizes monomeric as well as filamentous actin. (Melak et al., 2017). These tools for live-actin detection will allow us to monitor actin organization and dynamics at the onset of hypoxia and during reoxygenation in living cells

Investigating the influence of actin dynamics on gene expression at the onset of hypoxia

Nuclear actin has been shown to enhance transcription by associating with RNA Pol II. Thus, we speculate, that global transcription is repressed at very early stages of hypoxia due to shortage of nuclear actin. To test for productive transcription at the early onset of hypoxia the active form of RNAPII phosphorylated on Ser2 will be investigated by immunofluorescence. To analyze the abundance and size of active RNA Pol ll clusters and their relation to nuclear actin in normoxia, early hypoxia and during reoxygenation high resolution microscopy will be employed. Also, we will measure overall transcription rates across the genome in early hypoxia compared to normoxia using RNA Pol II Chip-Seq.

Exportin 6 (Exp6) mediates the nuclear export of profilin-actin complexes. Several studies have shown that the knock down of Exp6 results in nuclear actin accumulation (Stuven et al., 2003, Park et al., 2011). We will reduce Exp6 protein levels by lentiviral transduction of gene specific shRNA. Knockdown efficiency will be validated by qPCR and Western blotting. Exp6 knock down cells will allow us to analyze if changes in global transcription at the onset of hypoxia are dependent on nuclear actin deprivation as actin will be forced to remain nuclear in hypoxia.

Prof. Dr. med. Dörthe M. Katschinski
Speaker IRTG 1816
Heart Centre Göttingen, Department of Cardiovascular Physiology
+49 (0)551-39 9778 or 5896

Research interests: Hypoxia sensing, signalling and adaptation

Prof. Dr. Vsevolod V. Belousov
IRTG 1816 Mercator Fellow from Moscow State University, Russia
Shemyakin-Ovchinnikov Institute of bioorganic chemistry

Research interests: Biochemistry, redox engineering

Prof. Dr. Ajay M. Shah
Speaker British Heart Foundation Centre of Research Excellence
BHF Centre of Research Excellence, KCL

Research interests: NADPH oxidases, redox signalling and heart failure