Hypoxia regulates many hundreds of genes with important roles in ischemic and neoplastic disorders. Central to this response are the hypoxia inducible transcription factors (HIF). This work aimed to better understand the direct transcriptional response to HIF by mapping HIF-binding sites across the genome using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq). ChIP-seq for HIF in MCF-7 breast cancer cells under hypoxic conditions revealed more than 400 high-stringency HIF-binding sites genome-wide. Each member of the HIF heterodimer was present with near complete concordance. Binding of the two principle isoforms revealed a high degree of overlap with no differences in the DNA-binding motif. HIF-binding was associated with upregulation, but not downregulation of genes indicating that it functions as a transcriptional activator but not as a repressor. HIF-binding occurred preferentially at gene promoters, but was also present at promoter-distant sites, which were also associated with gene regulation, implicating long-range interactions in hypoxic gene activation. HIF-binding was associated with markers of open chromatin and active enhancers that were present in normoxia, indicating that HIF-binding sites are already “prepared” to bind HIF before the hypoxic stimulus. Analysis of normoxic and hypoxic RNA pol2 and H3K4me3 signals revealed distinctive hypoxia-inducible changes unique to HIF-binding genes. Comparable numbers of HIF-binding sites were observed in a second cell line (von Hippel-Lindau defective 786-O renal cancer cells) as in MCF-7 breast cancer cells, although approximately 65% were unique to 786-O cells. These unique sites were more frequently promoter-distant. Correlation with expression analyses from renal tumours indicated that many HIF-binding genes were upregulated in renal cancer. One such RCC unique promoter-distant HIF-binding site was identified at an intergenic locus on chromosome 11q13.3 that has been associated with renal cancer in Genome-Wide Association Studies. The HIF-binding site was in high linkage disequilibrium with the disease associated SNP and had the epigenetic hallmarks of an enhancer. Analysis of pan-genomic expression analyses identified the cell-cycle regulator cyclin D1 as highly HIF-regulated, and a physical association between the HIF-binding site and the CCND1 promoter could be determined. Furthermore, in a renal cancer cell line heterozygous at this locus, the RCC-protective allele disrupted HIF-binding leading to an allelic imbalance in cyclin D1 expression.

<p>Stroke is the third leading cause of death and the major course of long-term disabilities in industrialized countries. Most surviving stroke patients show some degree of spontaneous recovery, but persistent symptoms in sensorimotor and cognitive functions are common. The symptoms can be reproduced in experimental stroke models in rats by occlusion of the middle cerebral artery. Housing rats in an enriched environment (EE), i.e. group housing in a large cage with toys that are changed daily, increases neuronal plasticity in healthy rats and can also improve functional recovery after experimental stroke. </p><p> The present thesis investigates the effect of EE on the recovery of sensorimotor and cognitive functions one month after focal cerebral ischemia in rats, with emphasis on the underlying molecular mechanisms. Furthermore, EE-induced effect on gene expression in healthy rats was investigated after different periods of EE-housing and at different time points of the day. </p><p> We show an improved recovery of both sensorimotor and cognitive functions in rats housed in EE for one month after focal cerebral ischemia. The recovery of sensorimotor function correlated significantly to mRNA expression of the plasticity associated transcription factors NGFI-A and NGFI-B in hippocampus and cortical regions outside the infarct. Social interaction seems to be an important component for the beneficial effects of EE after focal cerebral ischemia. Microarray analysis of hippocampal gene expression after one month of postischemic environmental enrichment revealed no confirmable EE-induced changes that could explain the improved recovery in spatial memory. Interestingly, healthy rats housed in EE showed increased mRNA expression of NGFI-A and Krox-20 exclusively during the dark period of the day compared to rats housed in isolation. In addition, EE housed rats had a substantial diurnal variation in NGFI-A, Krox-20 and NGFI-B mRNA expression; this was absent in single-housed rats. EE-induced changes in gene expression are more evident during the dark period of the day, when rats are more active and can benefit from the stimulating environment. This is important to consider in future investigation of putative mediators of the EE-induced neuronal plasticity. </p><p> In summary, these findings may contribute to an increased understanding of the underlying molecular mechanisms behind improved functional recovery in rats housed in enriched environment after focal cerebral ischemia.</p>

Stroke is the third leading cause of death and the major course of long-term disabilities in industrialized countries. Most surviving stroke patients show some degree of spontaneous recovery, but persistent symptoms in sensorimotor and cognitive functions are common. The symptoms can be reproduced in experimental stroke models in rats by occlusion of the middle cerebral artery. Housing rats in an enriched environment (EE), i.e. group housing in a large cage with toys that are changed daily, increases neuronal plasticity in healthy rats and can also improve functional recovery after experimental stroke. The present thesis investigates the effect of EE on the recovery of sensorimotor and cognitive functions one month after focal cerebral ischemia in rats, with emphasis on the underlying molecular mechanisms. Furthermore, EE-induced effect on gene expression in healthy rats was investigated after different periods of EE-housing and at different time points of the day. We show an improved recovery of both sensorimotor and cognitive functions in rats housed in EE for one month after focal cerebral ischemia. The recovery of sensorimotor function correlated significantly to mRNA expression of the plasticity associated transcription factors NGFI-A and NGFI-B in hippocampus and cortical regions outside the infarct. Social interaction seems to be an important component for the beneficial effects of EE after focal cerebral ischemia. Microarray analysis of hippocampal gene expression after one month of postischemic environmental enrichment revealed no confirmable EE-induced changes that could explain the improved recovery in spatial memory. Interestingly, healthy rats housed in EE showed increased mRNA expression of NGFI-A and Krox-20 exclusively during the dark period of the day compared to rats housed in isolation. In addition, EE housed rats had a substantial diurnal variation in NGFI-A, Krox-20 and NGFI-B mRNA expression; this was absent in single-housed rats. EE-induced changes in gene expression are more evident during the dark period of the day, when rats are more active and can benefit from the stimulating environment. This is important to consider in future investigation of putative mediators of the EE-induced neuronal plasticity. In summary, these findings may contribute to an increased understanding of the underlying molecular mechanisms behind improved functional recovery in rats housed in enriched environment after focal cerebral ischemia.