Dynamic site-specific recruitment of RBP2 by pocket protein p130 modulates H3K4 methylation on E2F-responsive promoters

Abstract

The Histone 3 lysine 4 methylation (H3K4me3) mark closely correlates with active transcription. E2F-responsive promoters display dynamic changes in H3K4 methylation during the course of cell cycle progression. However, how and when these marks are reset, is not known. Here we show that the retinoblastoma binding protein RBP2/KDM5A, capable of removing tri-methylation marks on H3K4, associates with the E2F4 transcription factor via the pocket protein—p130—in a cell-cycle-stage specific manner. The association of RBP2 with p130 is LxCxE motif dependent. RNAi experiments reveal that p130 recruits RBP2 to E2F-responsive promoters in early G1 phase to bring about H3K4 demethylation and gene repression. A point mutation in LxCxE motif of RBP2 renders it incapable of p130-interaction and hence, repression of E2F-regulated gene promoters. We also examine how RBP2 may be recruited to non-E2F responsive promoters. Our studies provide insight into how the chromatin landscape needs to be adjusted rapidly and periodically during cell-cycle progression, concomitantly with temporal transcription, to bring about expression/repression of specific gene sets.

The emerging role of KDM5A in human cancer

“…Thus it may be feasible for an HDAC1 inhibitor to be applied synergistically with KDM5A inhibitors to weaken the viability of cancer cells [ 138 ]. Other modulators of such as EZH2, E2F4, and small ubiquitin-like modifier 2 may also be feasible for combination with KDM5A inhibitors for cancer therapy [ 70 , 80 , 139 ].…”

Physical association of functionally antagonistic enzymes: KDM5A interacts with MLLs to regulate gene expression in a promoter specific manner facilitating EMT and pluripotency

“…GST-tagged KDM5A deletion constructs (D1-D5; borrowed from Dr. Shweta Tyagi) (26) were expressed in BL21-DE3 and induced using 0.1-0.2 mM IPTG for 8hrs at 22 o C. The cell pellet collected was lysed in lysis buffer (26) and sonicated, incubated with glutathione agarose beads (sigma-G4510) at 4 o C for 4-6hrs. following protein quantification by SDS-PAGE, GST-bound protein was incubated with equal amount of whole cell lysates from U87MG and HaCaT for 6-8hrs.…”

“…MLL2 binds to the catalytic domain and PHD2 domain of KDM5A Further, to map the exact region of KDM5A that interacted with MLL2, pull-down experiments were setup with GST-fusion fragments of KDM5A which were overexpressed in BL21-DE3, purified using glutathione-GST beads, and used to pulldown endogenous protein from whole cell extracts of U87MG and HaCaT cells. Five deletion constructs of KDM5Awere used (D1-D5; where D1 denotes JmjN and ARID domain; D2 -PHD1-JmjC-ZF; D3 -PLU-1 like; D4 -PHD2; and D5 -PHD3 -borrowed from Dr. Shweta Tyagi (Zargar et al, (2018)). Both fragment D2 and D4 interacted with MLL2, in both the cell lines (figure 8C and supplementary figure S12).…”

KDM5 histone-demethylases contribute to replication stress response and tolerance

“…Whereas its role in the stable repression of E2F dependent genes has been well documented, whether it is also important for the regulation of the oscillation of E2F genes transcription during the cell cycle, is less known. However, a recent report describes its interaction with p130 in order to demethylate H3K4 on E2F promoters in G0 and early G1 [10].KDM5A can also act as a transcriptional activator. When enriched in gene bodies, it plays a role in the elongation step of Polymerase II (Pol-II) by maintaining low levels of H3K4 methylation [11].…”

“…Whereas its role in the stable repression of E2F dependent genes has been well documented, whether it is also important for the regulation of the oscillation of E2F genes transcription during the cell cycle, is less known. However, a recent report describes its interaction with p130 in order to demethylate H3K4 on E2F promoters in G0 and early G1 [10].…”