JHDM3A Module as an Effector Molecule in Guide-directed Modification of Target Chromatin

Abstract

With the objective of returning cells to their undifferentiated state through alteration of epigenetic states, small molecules have been used that specifically inhibit proteins involved in sustaining the epigenetic system. However, this chemicalbased approach can cause chaotic epigenomic states due to random actions of the inhibitors. We investigated whether JHDM3A/JMJD2A, a trimethylated histone H3-lysine 9 (H3K9me3)-specific demethylase, could function as an effector molecule to selectively demethylate target chromatin, with the aid of a guide protein to serve as a delivery vehicle. JHDM3A, which normally locates in euchromatin, spread out to heterochromatin when it was fused to heterochromatin protein-1␣ (HP1␣) or HP1␤; in these cells, demethylation efficiency was also markedly increased. A widely accepted hypothesis about the origins of cancer proposes that it is caused, at least in part, by the silencing of genes implicated in cell death and differentiation (1). Similarly, induced pluripotent stem cells (iPSCs) 2 (2) and somatic nuclear transfer clones are preceded by derepression of groups of genes responsible for stem cell properties and dedifferentiation, which are otherwise tightly suppressed in somatic cells (3). Fundamental changes in the nature of cells generally stem from perturbations in the transcriptional regulatory machinery; for example, changes in DNA methylation states at the regulatory DNA regions, mutations in chromatin remodeling complexes such as SWI/SNF, and/or changes in post-translational modifications of histone tails (4). Attempts to modulate DNA methylation by DNA methyltransferase inhibitors (5, 6), histone deacetylation by HDAC inhibitors (7-9), and histone methylation by histone demethylase inhibitors (10, 11) have received a great deal of attention. However, treating cells with small molecules such as these can lead to total inactivation of the target proteins, which could cause a chaotic alteration of epigenetic states by influencing the chromatin in its entirety and driving it to a structurally and functionally disorganized state. To avoid this, the epigenetic state of chromatin must be controlled at a local level. One means of achieving this is by targeting proteins that affect chromatin structure to specific genomic loci.Histone lysine demethylases (12) have roles in transcriptional regulation, cancer cell proliferation (13), and reprogramming of differentiated cells (14); this suggests that they could be used to alter the histone methylation states of specific chromatin regions. In an attempt to selectively modulate the epigenetic states of target loci, we tested the ability of histone H3-lysine 9 (H3K9)-specific demethylase JHDM3A/ JMJD2A (15, 16) to function as an effector molecule, together with various guide proteins that served as delivery vehicles to targeted chromatin regions. EXPERIMENTAL PROCEDURESCell Culture and Transfection-NIH3T3 and HEK293T cells were cultured in DMEM (Invitrogen) with 10% heatinactivated fetal bovine serum (Invitrogen) and 50 g/ml of streptom...

CTCF and Its Multi-Partner Network for Chromatin Regulation

“…Another CTCF histone demethylase partner is KDM4A. The first clue that CTCF could be a KDM4A partner was reported in 2011 by Kang's group, who performed transfection and immunofluorescence assays and observed that the demethylation frequency of KDM4A was enhanced by the presence of CTCF [102]. This study opened the window to another report in 2018, where co-immunoprecipitation was used to demonstrate that CTCF and KDM4A form a protein complex.…”

CTCF-KDM4A complex correlates with histone modifications that negatively regulate CHD5 gene expression in cancer cell lines

“…Given that CTCF plays a major role in the demethylation function of KDM4A [ 8 ], we decided to characterize its expression in our cellular model. By RT-qPCR, we observed that CTCF was overexpressed in the MCF7, MDA-MB-231 and HeLa cell lines when compared to the MCF 10A cells (Figure 3A ).…”

“…In vitro assays have reported that the presence of CTCF increases the demethylation frequency of KDM4A by up to 80%, suggesting that CTCF has a role in the demethylation function of KDM4A [ 8 ]. Additional support for these datasets was provided by another study that demonstrated that CTCF can interact with the KDM5B histone demethylase and increase its demethylation activity in breast cancer cell lines [ 33 ].…”

“…Neither the mechanism by which KDM4A negatively regulates CHD5 nor the mechanism by which KDM4A is recruited to this target site are known. Furthermore, in vitro assays have shown that the demethylation frequency of KDM4A increases up to 80% in the presence of the architectural protein CTCF [ 8 ], suggesting that CTCF may play a major role in the activity of KDM4A which has not been addressed until now. Hence, the aim of this study was to elucidate the mechanism underlying the role of CTCF and KDM4A on histone modifications and in the downregulation of CHD5 .…”

Quantifying difference in gene expression profile between bovine blastocysts derived by in vitro fertilization and somatic cell nuclear transfer

“…S1. The AxioVision intensity profile tool (v4.8) was used (Jeong et al, 2011). IVF, blastocysts from in vitro-fertilization; cSCNT and fSCNT, cumulus cell-and ear-skin fibroblast-derived somatic cell nuclear transfer blastocysts.…”