Epigenetic Regulation Of The HIF Pathway And Novel Therapeutic Opportunities
Abstract
The research presented in this dissertation is aimed at understanding the epigenetic
regulation of the HIF pathway and the efficacy of treatment for retinal diseases and
hemangioblastomas using HIF pathway inhibitors. Jumonji domain containing proteins
demethylate tri- and dimethylated histone lysines and regulate chromatin structure.
Substrate specificity of Jumonji domain-2 family histone demethylases (JMJD2A-C)
was evaluated. A candidate substrate-based approach demonstrated that JMJD2A-C
demethylate trimethylated lysine containing peptides from WIZ, CDYL1, CSB and G9a
proteins, all constituents of transcription repression complexes. Also preference of Arg
at –1 position and a smaller amino acid at –2 position was identified. The results
obtained are consistent with lax substrate specificities observed for the iron (II), 2-
oxoglutarate-dependent dioxygenases, and shed new light on the role of JMJD2 family
of KDMs during epigenetic transcriptional regulation.
Hypoxia inducible factor (HIF) plays a critical role in cellular adaptation to
hypoxia. Little is known about the epigenetic regulations during HIF-mediated
transcription of pro-angiogenic genes in oxygen-dependent retinal diseases and
hemangioblastomas. Hypoxic induction of a number of histone lysine demethylases
(KDMs) and stem cell markers in retinal pigment epithelial cells and VHL‒/‒ renal cell carcinoma cells was evaluated. Also, expression of pro-angiogenic genes (ADM,
GDF15, HMOX1, SERPE1 and SERPB8) was determined to be dependent on KDMs
under hypoxia. Further, treating cells with a general KDM inhibitor blocked the
induction of these pro-angiogenic genes. Expression of stem cell markers in
hemangioblastomas was determined to be due to activation of the HIF pathway. Further,
honokiol, digoxin, and doxorubicin, three recently identified HIF inhibitors from natural
sources effectively blocked the expression of stem cell markers. These results present
the possible cytological origin of neoplastic stromal cells in hemangioblastomas. Based
on our results we believe that inhibition of the HIF pathway is an attractive strategy for
the treatment of hemangioblastomas and retinal diseases. Also, blocking of KDMs
using specific inhibitors can inhibit the amplification of the HIF pathway.
TET2 is a Fe(II), 2OG dependent dioxygenase which can demethylate 5mC in a
CpG context, a key epigenetic modification. In this study we developed a HPLC based
analytical method for evaluating the catalysis by recombinant TET2 protein
Table of Contents
Introduction -- characterization of JMJD2 (Jumonji domain containing) family of histone demethylases -- Evaluating the role of histone demethylases in oxygen mediated pathophysiological conditions -- Development of a HPLC based method for evaluating the catalysis of TET2 DNA demethylase -- Conclusion ad future perspectives -- Appendix
Degree
Ph.D.