Drosophila p53 Is Required to Increase the Levels of the dKDM4B Demethylase after UV-induced DNA Damage to Demethylate Histone H3 Lysine 9

Abstract

Chromatin undergoes a variety of changes in response to UVinduced DNA damage, including histone acetylation. In human and Drosophila cells, this response is affected by mutations in the tumor suppressor p53. In this work, we report that there is a global decrease in trimethylated Lys-9 in histone H3 (H3K9me3) in salivary gland cells in wild type flies in response to UV irradiation. In contrast, flies with mutations in the Dmp53 gene have reduced basal levels of H3K9me3, which are then increased after UV irradiation. The reduction of H3K9me3 in response to DNA damage occurs preferentially in heterochromatin. Our experiments demonstrate that UV irradiation enhances the levels of Lys-9 demethylase (dKDM4B) transcript and protein in wild type flies, but not in Dmp53 mutant flies. Dmp53 binds to a DNA element in the dKdm4B gene as a response to UV irradiation. Furthermore, heterozygous mutants for the dKdm4B gene are more sensitive to UV irradiation; they are deficient in the removal of cyclobutane-pyrimidine dimers, and the decrease of H3K9me3 levels following DNA damage is not observed in dKdm4B mutant flies. We propose that in response to UV irradiation, Dmp53 enhances the expression of the dKDM4B histone demethylase, which demethylates H3K9me3 preferentially in heterochromatin regions. This mechanism appears to be essential for the proper function of the nucleotide excision repair system.In eukaryotic cells, the dynamics of chromatin structure play a central role in all processes involving nuclear DNA, including transcription, replication, recombination, and repair. It has been shown that ATP-dependent complexes that alter the structure and array of nucleosomes, together with complexes containing enzymes that modify different histone residues, are the primary modulators of chromatin structure.The most extensively studied histone modifications include acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and ADP-ribosylation (1, 2). It is possible to correlate the status of the chromatin with the residue that is modified and the type of modification, in a manner that is dependent on the specific histone involved. Some histone modifications are prevalent in heterochromatic regions, and others are preferentially linked to euchromatin. For instance, in the case of DNA transcription and replication, it is generally accepted that relaxed chromatin facilitates the incorporation of factors that recognize elements in the DNA, allowing multisubunit complexes to assemble and achieve these functions. A similar situation may occur during DNA repair, because the DNA repair machinery has to repair DNA in the context of chromatin (3).DNA damage by UV irradiation in eukaryotic cells is repaired via the nucleotide excision repair (NER) 3 mechanism. In vitro reconstituted assays have demonstrated that removal of a lesion requires recognition by XPC-HR23b and subsequent unwinding of the DNA duplex by TFIIH (3, 4). The resulting single strands of DNA are then stabilized by xeroderma pigmentosum A and replication pro...

The genetic depletion or the triptolide inhibition of TFIIH in p53 deficient cells induce a JNK-dependent cell death in Drosophila

“…Total extracts from salivary glands was performed as described previously (Palomera-Sanchez et al, 2010). Gels were transferred into nitrocellulose membranes and were revealed for western blot using an ECL chemiluminescence detection kit (Amersham).…”

PARP1-dependent recruitment of KDM4D histone demethylase to DNA damage sites promotes double-strand break repair

“…Remarkably, members of the KDM4 family are up-regulated in different types of human cancer, and, importantly, their depletion impairs cancer-cell proliferation and tumor formation (18)(19)(20)(22)(23)(24)(25)(26)(27). Moreover, several observations implicating histone demethylation in DDR have been recently reported (19,28,29). Of particular interest, it was recently shown that, in response to DNA damage, KDM4A and KDM4B undergo rapid degradation that facilitates foci formation of tumor protein P53 binding protein (53BP1) (30).…”

The emerging role of lysine demethylases in DNA damage response: dissecting the recruitment mode of KDM4D/JMJD2D to DNA damage sites

“…57 Previous work has also implicated the drosophila KDM4B in both UV-and g irradiationinduced DNA damage. 56 They showed that upon exposing drosophila salivary gland cells to UV irradiation, KDM4B protein is upregulated in a p53-dependent manner and this was accompanied by a decrease in H3K9me3 levels, which occurs preferentially in heterochromatin. Importantly, drosophila flies heterozygous for KDM4B mutant are more sensitive to UV irradiation and are deficient in the removal of Cyclobutane-PyrimidineDimers (CPDs) from damage sites.…”

“…In support of this, a decrease in H3K9me3 levels, which occurs preferentially at heterochromatic chromocenter regions, was documented in drosophila. 56 Moreover, decrease in the levels of H3K9me2/3 at gH2AX-positive regions was reported at 20 minutes after IR in mammalian cells. 87 Similarly, Young and colleagues observed local reduction in H3K9me3 levels at sites of damage in cells expressing low to moderate levels of EGFP-KDM4B fusion.…”

“…The JmjC catalytic domain forms an enzymatically active pocket that coordinates the 2 co-factors, ferrous oxide (Fe(II)) and a-ketoglutarate, that are needed for the radical-based oxidative demethylation reaction (reviewed in [44][45][46][47] ). KDM proteins are involved in a plethora of cellular processes including gene expression regulation, [48][49][50][51][52][53] DNA replication, 41,54 DNA damage response, [55][56][57][58] worm development and germ cell apoptosis, 59 cell differentiation and renewal of embryonic stem cells. 60 Interestingly, several KDM proteins show oncogenic activity and are overexpressed in various types of human cancer (reviewed in 41,61 ).…”