UHRF2 Interacts With TIP60 And Regulates H3K9ac And H3K14ac Via RING Finger Domain

BACKGROUNDUHRF2 is a multi-domain nucleoprotein with E3 ubiquitin ligase activity.Involved in cell cycle,DNA damage repair,epigenetics and ubiquitin-proteasome system.In our previous work,our team confirmed that UHRF2 was involved in the regulation of HBV transcription and replication and regulated the level of histone H3 acetylation with cccDNA binding.Recent studies have shown that UHRF2 interacts with histone H3K9 ac and H3K14 ac.Based on the above research results,this paper will continue to explore the regulatory mechanism between UHRF2 and histone acetylation.METHODThis paper mainly through western blot to determine the effect of UHRF2 on the regulation of H3K9 ac,H3K14ac,TIP60,H2AK5 ac and HDAC1.The interaction between UHRF2 and TIP60 and HDAC1 was verified by Co-IP experiment.The relationship between UHRF2 and TIP60 and HDAC1 was verified by IF experiment.The half-life experiment was performed to verify the regulation of UHRF2 on the half-life of TIP60.In vivo ubiquitination experiments verify the ubiquitination relationship of UHRF2 to TIP60.Immunohistochemical was used to test the relationship between UHRF2 and H3K9 ac,H3K14ac,TIP60 and H2AK5 a.RESULT1)Western blot showed that high expression of UHRF2 in normal cells could up-regulate the expression of H3K9 ac and H3K14 ac,while the expression of UHRF2 in tumor cells inhibited the expression of H3K9 ac and H3K14 ac.Interference UHRF2 is consistent with the over-expression of UHRF2.2)Western blot experiments confirmed that deletion of UHRF2 YDG domain or RING domain in HEK293,LO2 and HepG2 cells could abolish the regulatory effect of UHRF2 on H3K9 ac and H3K14 ac.3)Co-IP and IF experiments confirmed that UHRF2 and TIP60 and HDAC1 co-localization and binding.The YDG domain,PHD domain and RING domain of UHRF2 is essential for the binding of UHRF2 to TIP60.Lack of these domains can significantly reduce the binding of UHRF2 to TIP60,while the combination of UHRF2 and HDAC1 has no significant effect.4)Western blot showed that UHRF2 could up-regulate the expression and activity of TIP60 in normal cells,while the expression of UHRF2 in tumor cells could inhibit the expression of TIP60 and the activity of TIP60,and had no significant effect on HDAC1 expression.And the regulatory effect of UHRF2 to TIP60 was abolished with the proteasome inhibitor MG132.5)Half-life experiments confirmed that overexpression of UHRF2 in normal cells(HEK293 and LO2 cells)could prolong the half-life of TIP60,whereas over-expression of UHRF2 in tumor cells(HepG2 cells)could accelerate the degradation rate of TIP60,and lack of the RING domain of UHRF2 can abolish this regulatory.6)In vivo ubiquitination experiments confirmed that overexpression of UHRF2 in normal cells(HEK293 and LO2 cells)increased ubiquitination of TIP60,whereas overexpression of UHRF2 in tumor cells(HepG2 cells)inhibited the ubiquitination of TIP60.7)Western blot showed that TIP60 could upregulate the expression of H3K9 ac and H3K14 ac in HEK293,LO2 cells and HepG2 cells,and the expression of H3K9 ac and H3K14 ac could be inhibited when TIP60 was distzurbed.8)Western blot showed that TIP60 could inhibit the expression of H3K9 ac and H3K14 ac by over-expressing UHRF2 and inhibiting TIP60,and the expression of H3K9 ac and H3K14 ac could be further up-regulated by UHRF2 and TIP60.When we applied TIP60 inhibitor MG149 Cells,both the overexpression of wild-type UHRF2 or UHRF2 domain mutants can not continue to control H3K9 ac and H3K14 ac.9)TIP60,H2AK5 ac,H3K9ac and H3K14 ac showed low expression in UHRF2 overexpressing hepatocellular carcinoma tissues,while the high expression of UHRF2 in adjacent tissues was accompanied by high TIP60,H2AK5 ac,H3K9ac and H3K14 ac expression.CONCLUSIONIn normal cells,UHRF2 up-regulated the protein expression and enzyme activity of TIP60,and up-regulated H3K9 ac and H3K14 ac.In tumor cells,UHRF2 inhibited the protein expression and enzyme activity of TIP60,and inhibited the expression of H3K9 ac and H3K14 ac.