Biochemical and Biophysical Research Communications
Plant homologs of mammalian MBT-domain protein-regulated KDM1 histone lysine demethylases do not interact with plant Tudor/PWWP/MBT-domain proteins
Introduction
Post-translational histone modifications, e.g., acetylation, methylation, and ubiquitination, play central role in gene regulation in all eukaryotic organisms, determining the active or inactive state of the chromatin. These modifications are effected by diverse histone-modifying enzymes, such as histone deacetylases, histone lysine demethylases, histone methyltransferases, and histone deubiquitinases. Among those, LSD1/KDM1-type histone lysine demethylases [1] represent one of the most recently discovered [2], yet clearly central factors in controlling chromatin in different organisms, from animals to plants [3], [4], [5]. In animal calls, LSD1/KDM1A promotes demethylation of dimethylated lysine 4 (K4) of histone H3 [2] and often functions as negative regulator of gene expression [6]. LSD1/KDM1A usually acts in complex with CoREST that modulates the gene repression function of LSD1 in vivo, a histone methyltransferase (HMT; e.g., G9a), HDAC1/2 histone deacetylases, and DNA binding zinc finger proteins (e.g., either REST or ZNF217) [2], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. However, none of these components recognize methylated histones that represent the direct substrates of LSD1/KDM1A; recently, this role has been attributed to the SFMBT1 [Scm (Sex comb on midleg) with four MBT (malignant brain tumor) domains 1] protein, which functions as part of the LSD1/KDM1A-based repressor complex and is known to bind different forms of methylated histones [18], [19].
In plants, our knowledge of LSD1/KDM1 complexes is just emerging. For example, Arabidopsis, one of the major model plants, encodes four KDM1 proteins, KDM1A (FLD), KDM1B, KDM1C, and KDM1D [1]. KDM1C has been shown to interact with histone methyltransferase SURV5 [20], [21], [22] and histone deubiquitinase OTLD1 [23]. MBT domains are conserved within proteins from different organisms, from animals to insects to plants [24], [25]. Thus, we examined whether Arabidopsis LSD1/KDM1A-type histone lysine demethylases can recognize plant Tudor/PWWP/MBT-domain proteins [24], [25], i.e., the Arabidopsis SFMBT1-like proteins (SLs). Our data indicate no such interactions and suggest that plant LSD1/KDM1 histone lysine demethylases are directed to their substrates by a mechanism different from their mammalian counterparts.
Section snippets
Plasmid construction
The coding sequences of SL1, SL2, SL3, and SL4, KDM1C and FLD were amplified from Arabidopsis thaliana cDNA library using primers detailed in Table S1. The SL5 sequence failed to amplify and was not examined in this work. For transient expression in Nicotiana benthamiana, the amplified SL1, SL2, SL3, and SL4 as well as SURV5 [20] were cloned into the HindII-SalI, EcoRI-SalI, XhoI-SalI, SalI-SacII, and SalI sites of pSAT4-nEYFP [26], respectively, resulting in pSAT4-SL1-nYFP, pSAT4-SL2-nYFP,
Arabidopsis SL proteins
Amino acid sequence analysis identified five relatively close homologs of the human SFMBT1 protein encoded by the Arabidopsis genome. These sequences were aligned with SFMBT1 by CLC Main Workbench 7.6.4 (http://www.clcbio.com) software, using default parameters (Fig. S1), and their phylogenetic tree was generated (Fig. 1). All of these five genes, i.e., SL1 (At1g51745), SL2 (At1g80810), SL3 (At2g48160), SL4 (At3g63070), and SL5 (At5g08230) belong to the Tudor domain “Royal family”, which
Acknowledgments
I.S. was supported in part by the Fulbright Fellowship. The work in the V.C. laboratory is supported by grants from NIH (R01 GM50224), NSF (MCB 1118491), USDA/NIFA (2013-02918), BARD (IS-4605-13C), and BSF (2011070) to V.C.
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