Plant homologs of mammalian MBT-domain protein-regulated KDM1 histone lysine demethylases do not interact with plant Tudor/PWWP/MBT-domain proteins

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Highlights

  • Interaction of plant SFMBT1-like proteins with KDM1 histone demethylases was tested.

  • Four Arabidopsis homologs of the mammalian SFMBT1 were selected for study.

  • Two Arabidopsis KDM1 histone demethylases, KDM1C and FLD, were tested.

  • None of the Arabidopsis SFMBT1 homologs showed interaction with KDM1C or FLD.

  • Plants and animals may use different mechanisms to direct KDM1 to methyl-histones.

Abstract

Histone lysine demethylases of the LSD1/KDM1 family play important roles in epigenetic regulation of eukaryotic chromatin, and they are conserved between plants and animals. Mammalian LSD1 is thought to be targeted to its substrates, i.e., methylated histones, by an MBT-domain protein SFMBT1 that represents a component of the LSD1-based repressor complex and binds methylated histones. Because MBT-domain proteins are conserved between different organisms, from animals to plants, we examined whether the KDM1-type histone lysine demethylases KDM1C and FLD of Arabidopsis interact with the Arabidopsis Tudor/PWWP/MBT-domain SFMBT1-like proteins SL1, SL2, SL3, and SL4. No such interaction was detected using the bimolecular fluorescence complementation assay in living plant cells. Thus, plants most likely direct their KDM1 chromatin-modifying enzymes to methylated histones of the target chromatin by a mechanism different from that employed by the mammalian cells.

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.

References (33)

  • A. Krichevsky et al.

    C2H2 zinc finger-SET histone methyltransferase is a plant-specific chromatin modifier

    Dev. Biol.

    (2007)
  • S. Maurer-Stroh et al.

    The Tudor domain “Royal Family”: Tudor, plant Agenet, Chromo, PWWP and MBT domains

    Trends biochem. Sci.

    (2003)
  • V. Citovsky et al.

    Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta

    J. Mol. Biol.

    (2006)
  • C.D. Hu et al.

    Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation

    Mol. Cell

    (2002)
  • S.F. Altschul et al.

    Basic local alignment tool

    J. Mol. Biol.

    (1990)
  • X. Zhou et al.

    Evolutionary history of histone demethylase families: distinct evolutionary patterns suggest functional divergence

    BMC Evol. Biol.

    (2008)
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