טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentNadar Ponniah Prathamesh T.
SubjectLinking Histone Demethylation with RNA:
Studying the Biological Functions of KDM4D-RNA
Interactions
DepartmentDepartment of Biology
Supervisor Professor Nabieh Ayoub
Full Thesis textFull thesis text - English Version


Abstract

Chromatin, the repeating unit of DNA wrapped around core histone proteins, is the physiological template of our genome and is packaged into a less understood higher order structure. This structure is greatly influenced by multiple factors such as chromatin modifying enzymes and over 11 types of post-translational modifications (PTMs). Histones are subjected to a variety of PTMs including lysine methylation, which is one of the most common modifications of the histone tails. Methylation causes conformational change in chromatin making it accessible or inaccessible to transcriptional machinery. Additionally, methylation of lysine residues is a dynamic modification that regulates the activity and the function of histone and non-histone proteins.

KDM4A-E (also known as JMJD2A-E) family consists of five members which specifically catalyses the demethylation of H3K9me2/me3, H3K36me2/me3, H1.4K26me2/me3 and H3K56me3. KDM4A-E family is implicated in multiple cellular processes critical to carcinogenesis, including transcription, DNA damage response, cell-cycle regulation, cellular differentiation and senescence. The mechanisms by which KDM4A-E members regulate these diverse processes are unclear mainly because, their biology is largely unknown. Moreover, even though KDM4A-E proteins erase the same histone methylation marks of histone H3, they do not have redundant functions.

KDM4D is a histone demethylase that removes tri- and di- methyl residues from H3K9 and is involved in transcriptional regulation and carcinogenesis. We recently showed that KDM4D is recruited to DNA damage sites and promotes double-strand break repair. It remains unknown how KDM4D binds its histone targets and specifically how it is recruited to DNA damage sites. We hypothesize that RNA molecules mediate KDM4D recruitment to its genomic binding sites. In support of our hypothesis, we show that KDM4D binds RNA in vitro and in vivo. We found that KDM4D-RNA interaction is sequence independent but length dependent. Moreover, we demonstrated that KDM4D interaction with RNA is independent of its demethylase activity. Next, we mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 350 to 432 of KDM4D. In addition, we demonstrated that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. Interestingly, KDM4D-RNA interaction is also required for KDM4D accumulation at DNA breakage sites.

Collectively, this study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin. Also, it implicates RNA molecule in the DNA damage response by regulating KDM4D to DNA damage sites. Our data therefore shed mechanistic insights into the mechanism(s) that regulate the recruitment of KDM4D histone demethylases to its target genes. In addition, it revealed new pathways that links RNAs and epigenetic regulation of gene activity.