A developmental genetic analysis of the lysine demethylase KDM2 mutations in Drosophila melanogaster

Abstract

Post-translational modification of histones plays essential roles in the transcriptional regulation of genes in eukaryotes. Methylation on basic residues of histones is regulated by histone methyltransferases and histone demethylases, and misregulation of these enzymes has been linked to a range of diseases such as cancer. Histone lysine demethylase 2 (KDM2) family proteins have been shown to either promote or suppress tumorigenesis in different human malignancies. However, the roles and regulation of KDM2 in development are poorly understood, and the exact roles of KDM2 in regulating demethylation remain controversial. Since KDM2 proteins are highly conserved in multicellular animals, we analyzed the KDM2 ortholog in Drosophila. We have observed that dKDM2 is a nuclear protein and its level fluctuates during fly development. We generated three deficiency lines that disrupt the dKdm2 locus, and together with 10 transposon insertion lines within the dKdm2 locus, we characterized the developmental defects of these alleles. The alleles of dKdm2 define three phenotypic classes, and the intragenic complementation observed among these alleles and our subsequent analyses suggest that dKDM2 is not required for viability. In addition, loss of dKDM2 appears to have rather weak effects on histone H3 lysine 36 and 4 methylation (H3K36me and H3K4me) in the third instar wandering larvae, and we observed no effect on methylation of H3K9me2, H3K27me2 and H3K27me3 in dKdm2 mutants. Taken together, these genetic, molecular and biochemical analyses suggest that dKDM2 is not required for viability of flies, indicating that dKdm2 is likely redundant with other histone lysine demethylases in regulating normal development in Drosophila.

Differentiating Drosophila female germ cells initiate Polycomb silencing by altering PRC2 sampling

“…Because both PRC1 and PRC2 can directly interact with DNA and the histone modifications catalyzed by the other complex, each complex could putatively recruit the other or synergize to promote higher recruitment at overlapping sites. Flies have homologues of Pcl and Kdm2 proteins but fly Pcl lacks sequence specificity in vitro (Choi et al, 2017) and fly Kdm2 is dispensable for viability (Shalaby et al, 2017;Zheng et al, 2014). Furthermore, while PRC1-catalyzed H2AK119ub enriches PcG proteins in mouse and fly embryonic cell cultures, Cooper et al, 2014;Fursova et al, 2019;Kahn et al, 2016), it has a minimal effect on PRC2 localization and Polycomb silencing in many fly tissues (Gutiérrez et al, 2012;Pengelly et al, 2015).…”

Interdependence of PRC1 and PRC2 for recruitment to Polycomb Response Elements

“…dRAF was reported to produce the bulk of Drosophila H2AK118ub and mutations in the Kdm2 gene were said to enhance the homeotic phenotypes of heterozygous Pc mutants (15). However, a recent study suggests that Kdm2 is not essential for fly viability (17) questioning the importance of dRAF for PcG repression. Finally, Drosophila RING1 can potentially form a dimer with a third Drosophila PCGF protein called L(3)73Ah.…”

The lysine demethylase dKDM2 is non-essential for viability, but regulates circadian rhythms in Drosophila

“…Drosophila has 13 KDMs, with only one conserved KDM2 ortholog, dKdm2 (LAGAROU et al 2008;KAVI and BIRCHLER 2009;ZHENG et al 2014;HOLOWATYJ et al 2015). Based on analyses of 13 mutant alleles of dKdm2 gene, we have concluded that dKdm2 is not required for normal development in Drosophila (ZHENG et al 2014).…”

“…Drosophila has 13 KDMs, with only one conserved KDM2 ortholog, dKdm2 (LAGAROU et al 2008;KAVI and BIRCHLER 2009;ZHENG et al 2014;HOLOWATYJ et al 2015). Based on analyses of 13 mutant alleles of dKdm2 gene, we have concluded that dKdm2 is not required for normal development in Drosophila (ZHENG et al 2014). This notion is unexpected for the following reasons: first, dKdm2 has been identified as a subunit of the dRING-associated factor (dRAF) complex, a Polycomb group (PcG) complex that was proposed to play critical roles in coordinating trans-histone regulation by replacing the active H3K36me2 marker with a repressive mono-ubiquitinated H2A K118 (H2AK118ub or H2Aub) marker (LAGAROU et al 2008).…”

“…A major concern in our previous genetic analyses is that all existing mutant alleles of the dKdm2 locus have their limitations: the transposon insertion lines are either hypomorphic or fail to affect the mRNA and protein levels of dKDM2, while the three deficiency lines uncover the dKdm2 locus disrupt both dKdm2 and its neighboring genes (ZHENG et al 2014). Having a molecularly defined dKdm2 null allele that only disrupts the dKdm2 locus is thus required to resolve the aforedescribed contradictory observations, and will enable us to draw stronger conclusions about the dKdm2 null phenotypes and its molecular functions.…”