Structural And Functional Research Of Histone Demethylase JMJD5

Covalent modifications of core histones,including acetylation,methylation,ubiquitination and phosphorylation,have been confirmed to be involved in a variety of cellular processes in eukaryotes,including transcription regulation and chromatin dynamics.Histone lysine methylation is considered to be one of the most complicated modifications because its diverse functions depend on the sites and the degree of methylation.Histone lysine methylation is dynamically regulated by the interplay of histone methyltransferases with demethylases.There are two kinds of demethylases,including LSD1 family and proteins containing JmjC domain.The members of LSD1 family have monoamine oxidase activity.Proteins containing JmjC domain are members of 2-oxoglutarate oxygenases superfamily,which utilize Fe(II)and 2-oxoglutarate as co-factors to produce unmethylated lysine.JMJD5,also known as KDM8,is a newly identified JmjC domain-containing protein that shows histone demethylase activity towards H3K36me2 serving as a transcriptional activitor.JMJD5 are also related to other diverse cell processes,including cell cycle,cancer cell proliferation,embryonic growth and circadian systems.It is worth mentioning that JMJD5 has been identified as a hydroxylase in mice.JMJD5 functions as a novel osteoclastogenic repressor by inducing the degradation of NFATc1,a necessary and sufficient transcription factor for osteoclasogenesis,via its hydroxylase activity.In the context,we determined the crystal structure of the JmjC domain of JMJD5.Our structure revealed that the JmjC domain of JMJD5 adopts a conserved jelly-roll-like fold.However,three residues within the JmjC domain of JMJD5 were found to occupy the entrance to the active pocket,thereby preventing the binding of dimethylated lysine residues in comparison with the structures of PHF8 and ceKDM7A in complex with dimethylated histone substrates.Comparing the structure of JMJD5 with that of FIH indicated that JMJD5 might act as a protein hydroxylase.The biochemical assays reveal JMJD5 interacts with histone octamers directily.The binding of JMJD5 to the histone octamers indicated that the core histone octamers might be a potential substrate for JMJD5 to function as a protein hydroxylase.Our work provides new clues to the study of the function of JMJD5.Cenp-O complex are important components of CCAN.CCAN serves as a core structure on centromeric chromatin,which connects the centromeric DNA and the outer kinetochore.Cenp-O complex are not essential for the growth of DT40 cells because DT40 knockout cells for these proteins are viable.DT40 cells with a depletion of CENP-U are viable but show a slight mitotic delay.Co-expression of these five proteins was performed simultaneously in Escherichia coli which demonstrated that these proteins form a stable complex in vitro.Localization of CENP-O,-P,-Q,and-U is interdependent and CENP-R localization occurs downstream of these four proteins,suggesting that CENP-O,-P,-Q,and-U form a tight complex and that CENP-R associates with this complex.There are little knowledge about the interactions of the subunits.Therefore,Purification and even structural study of the complex will be necessary for the better understanding of the interactions of the subunits and kinetochore assembly.In this context,we expressed by insect cells and purified Cenp-O complexs containing four and five subunits,respectively.The results of the purification revealed the subunits of the complex tended to be degraded.Crystallization conditions of Cenp-O complex were screened.Unfortunately,we did not obtain any crystals for the complex.Our results give some basic information for the future research.