Gene duplications of JmjC-containing histone demethylases in angiosperms

47 Pages·2015·2.73 MB·English

Plant Physiology Preview. Published on June 9, 2015, as DOI:10.1104/pp.15.00520

1 Running head: Gene duplications of JmjC-containing histone demethylases in
2 angiosperms

3
4 Correspondence:
5 Hong Ma,
6 School of Life Sciences, Fudan University, Shanghai 200438, China
7 Tel: [phone],
8 Email: [email protected];
9
10 Liangsheng Zhang,
11 School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
12 Tel: [phone],
13 Email: [email protected]
14
15 Research area: Genes, Development and Evolution
16
17
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18 Expansion and functional divergence of JmjC-containing histone demethylases:
19 significance of duplications in ancestral angiosperms and vertebrates
20
21 Shengzhan Qian1, Yingxiang Wang1, Hong Ma1,2* and Liangsheng Zhang3,4*
22 1State Key Laboratory of Genetic Engineering and Collaborative Innovation Center of Genetics
23 and Development, Ministry of Education Key Laboratory of Biodiversity Science and Ecological
24 Engineering and Institute of Biodiversity Sciences, Institute of Plant Biology, Center for
25 Evolutionary Biology, School of Life Sciences, Fudan University, Shanghai 200438, China
26 2Institute of Biomedical Sciences, Fudan University, Shanghai 200032, China
27 3Department of Bioinformatics, School of Life Sciences and Technology, Tongji University,
28 Shanghai 200092, China
29 4Advanced Institute of Translational Medicine, Tongji University, Shanghai 200092, China

30
31 

32 One-sentence summaries:
33 Duplication and sequence divergence of a gene family for histone demethylases likely
34 contributed to the enhancement of chromatin-based regulation in angiosperms and vertebrates
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35 This work was supported by grants from the National Natural Science Foundation of China
36 (91131007) and the Ministry of Science and Technology of China (2011CB944603).
37 Address correspondence to [email protected] or [email protected].
38
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39 ABSTRACT
40 Histone modifications, such as methylation and demethylation, are crucial mechanisms altering
41 chromatin structure and gene expression. Recent biochemical and molecular studies have
42 uncovered a group of histone demethylases called Jumonji C (JmjC) domain proteins. However,
43 their evolutionary history and patterns have not been examined systematically. Here we report
44 extensive analyses of eukaryotic JmjC genes and define 14 subfamilies, including the KDM3,
45 KDM5, JMJD6, PKDM11 and PKDM13 subfamilies shared by plants, animals and fungi. Other
46 subfamilies are detected in plants and animals but not in fungi (PKDM12) or in animals and fungi
47 but not in plants (KDM2 and KDM4). PKDM7, PKDM8 and PKDM9 are plant-specific groups,
48 whereas JARID2, KDM6 and PKDM10 are animal specific. In addition to known domains, most
49 subfamilies have characteristic conserved amino acid motifs. Whole genome duplication (WGD)
50 was likely an important mechanism for JmjC duplications, with four pairs from an
51 angiosperm-wide WGD and others from subsequent WGDs. Vertebrates also experienced JmjC
52 duplications associated with the vertebrate ancestral WGDs, with additional mammalian paralogs
53 from tandem duplication and possible transposition. The sequences of paralogs have diverged in
54 both known functional domains and other regions, showing evidence of selection pressure. The
55 increases of JmjC copy number and the divergences in sequence and expression might have
56 contributed to the divergent functions of JmjC genes, allowing the angiosperms and vertebrates to
57 adapt to a great number of ecological niches and contributing to their evolutionary successes.
58
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59 Chromatin-based regulation is an important mechanism of modulating eukaryotic chromatin
60 structure and gene expression, by altering DNA and histone modifications rather than changing
61 DNA sequences. The eukaryotic chromatin contains a group of highly conserved proteins called
62 histones, including the core histones H2A, H2B, H3, and H4, as well as the linker histone H1.
63 The core histones, with two copies each, form an eight-subunit complex, which is wrapped by
64 146 base pairs of DNA to form the nucleosome, the basic unit of chromatin. In each nucleosome,
65 the hydrophobic carboxyl-terminal regions of the eight subunits occupy the interior, whereas the
66 hydrophilic amino-terminal regions (the tails) extend outward (Luger et al., 1997). The covalent
67 histone modifications occur on the tails of the core histone proteins and encode epigenetic
68 information that can be passed through mitosis, sometimes even meiosis, and alter chromatin
69 structure and modulate genomic functions. Specifically, histone modifications include
70 methylation, acetylation, phosphorylation, ubiquitination and sumoylation. Chromatin structure
71 can be regulated by three classes of proteins: DNA methylase and demethylases, chromatin
72 remodelers that regulate nucleosome positioning, and enzymes for histone modifications.
73 Among various histone modifications, the role of methylation varies in different species
74 (Feng et al., 2010; Liu et al., 2010) but is relatively stable and suited for the transmission of
75 epigenetic information (Strahl and Allis, 2000), sometimes even transgenerationally as supported
76 by a recent study in Arabidopsis (Arabidopsis thaliana) showing that the vernalized state can be
77 partially transgenerational inherited due to a defect in the reduction of H3K27me3 levels at the
78 FLC locus (Crevillen et al., 2014). Histone methylation occurs on arginine and lysine residues
79 and is involved in a wide range of biological processes including gene expression, chromatin
80 structure, dosage compensation, and epigenetic memory (Martin and Zhang, 2005). A lysine
81 residue can be mono-, di- or tri-methylated; however, an arginine residue can only be mono- and
82 di-methylated. Different histone modifications regulate distinct functional outcomes within an
83 epigenetic marking system: methylation at histone H3 lysine 4 (H3K4), H3K36 and H3K79 is
84 correlated with higher gene expression, whereas methylation at H3K9, H3K27 and H4K20 is
85 associated with lower gene expression, hence often termed ‘activating’ or ‘repressing’ marks,
86 respectively, although the causal relationships between histone modification and transcriptional
87 activity is still elusive (Martin and Zhang, 2005; Henikoff and Shilatifard, 2011; Dong and Weng,
88 2013).
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89 Histone methylation has been regarded as an irreversible modification for a long time,
90 because of the stable nature of the C-N bond; this idea is also supported by similar half-lives of
91 histone fractions and methyl lysine and methyl arginine marks (Byvoet et al., 1972). In 2004, the
92 discovery of the first histone demethylase, known as LSD1 (lysine specific demethylase 1),
93 provided experimental evidence for enzymatic demethylation (Shi et al., 2004). The single-copy
94 LSD1 mediates oxidative demethylation on mono- or di-methylated H3K4 and/or H3K9, but not
95 tri-methylated H3K4 (Shi et al., 2004). Subsequently, a second and larger class of demethylases
96 containing a JmjC domain was identified (Tsukada et al., 2006). Unlike LSD1, the JmjC proteins
97 do not require a protonated nitrogen and can also reverse tri-methylated histone lysine state (Shi
98 and Whetstine, 2007). Five amino acid residues within predicted cofactor binding sites in the
99 JmjC domain are conserved and important for enzymatic activity (Klose et al., 2006). Among
100 these, three residues bind to the Fe (II) cofactor and two other residues are required for
101 α-ketoglutarate (αKG) binding.
102 Several plant JmjC genes are known to have important functions in regulating development
103 and environmental responses (Liu et al., 2010; Chen et al., 2011; Kooistra and Helin, 2012). For
104 example, Polycomb Repressive Complex 2 (PRC2), a conserved and key transcriptional regulator
105 in animals and plants, has been demonstrated to have H3K27me3 affinity (Cao et al., 2002). In
106 Arabidopsis, REF6/AtPKDM9A and ELF6/AtPKDM9B are closely related JmjC paralogs and
107 have H3K27me3 demethylase activities (Lu et al., 2011; Crevillen et al., 2014), but have different
108 roles in the regulation of flowering time (Noh et al., 2004). The elf6 mutant flowers early and
109 exhibits reduced expression of the flowering repressor FLC (Noh et al., 2004), due to an elevated
110 level of the H3K27me3 at the FLC locus (Crevillen et al., 2014), but the ref6 mutant shows an
111 FLC- dependent late-flowering phenotype (Noh et al., 2004). In addition, a recent study showed
112 that loss of H3K27me3 was observed after salt priming in Arabidopsis seedlings (Sani et al.,
113 2013), suggesting that some JmjC genes may be induced upon salt stress. To date, many members
114 of JmjC gene family have not been characterized genetically or biochemically; nevertheless,
115 sequence and evolutionary analyses can help predict their functions in histone demethylation.
116 However, the expression and functional divergence of JmjC duplicates have not been studied
117 extensively, especially in response to abiotic stresses, such as ABA, salt and drought. In addition,
118 members of the PKDM7 subfamily exhibited distinct expression patterns correlated with their
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119 different enzymatic activities (Lu et al., 2008), but the underlying sequence divergence and
120 dynamic evolution are still unknown.
121 An early study of the JmjC gene family using sequences from Arabidopsis, rice and human
122 defined 12 subfamilies according to phylogenetic relationships, with support from the presence of
123 other domains (Zhou and Ma, 2008). These domains play potential regulatory roles in the
124 demethylation process, such as the recognition of methylated histone marks (e.g. PHD and Tudor),
125 protein-protein interaction (e.g. F-box) and DNA binding (e.g. C2H2 zinc finger) (Zhou and Ma,
126 2008). However, the previous reports have focused on a single or a few species (Lu et al., 2008;
127 Zhou and Ma, 2008), thus there have not been a systematic study of JmjC family in plants or
128 animals. The recent progresses in genome sequencing provide an opportunity to gain additional
129 insights into the evolution of the JmjC family during the histories of angiosperms and vertebrates.
130 Histone methylation mainly mediated by SET domain protein methyltransferase, and genome
131 duplication has resulted in an increase of SET copy numbers. In Arabidopsis, five duplicated gene
132 pairs are retained after recent genome duplication events, and nineteen pairs are retained in
133 Populus trichocarpa (poplar) (Lei et al., 2012). It would also be helpful to learn whether the
134 JmjC gene family has a similar evolutionary pattern to that of the SET genes. The evolutionary
135 patterns of these two families could inform the balanced influence of demethylation and
136 methylation on species evolution and divergence. In this study, we have characterized the
137 evolution of JmjC genes in major eukaryotic lineages, including land plants, using phylogenetic
138 and domain analyses, and define 14 monophyletic subfamilies: JARID2, JMJD6, KDM2, KDM3,
139 KDM4, KDM5, KDM6, PKDM7, PKDM8, PKDM9, PKDM10, PKDM11, PKDM12 and
140 PKDM13. We show that some families underwent distinct gene duplication during evolution in
141 angiosperms, especially in the ancestor of extent angiosperms.
142
143
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144 RESULTS
145 Identification of JmjC genes in plants, animals and fungi
146 The complete set of JmjC genes was identified from a comprehensive dataset that contains
147 selected plants, animals and fungi based on HMMER software. In all, 434 sequences, each
148 containing a JmjC domain, were retrieved from 35 different organisms including 11 plants, 16
149 metazoans, 7 fungi and Monosiga brevicollos (a unicellular choanoflagellate, a protist related to
150 animals) (Fig.1; Supplemental Tables S1 and S2). The identified JmjC proteins range in size from
151 266 to 2740 aa. Among major lineages of plants, JmjC genes are present in algae, Bryophyta,
152 Pteridophyta, and angiosperms. The copy number of JmjC genes varies considerably among
153 plants, ranging from 2 in the green algae Chlamydomonas reinhardtii and Volvox carteri to 17 in
154 rice O. sativa (monocot) and 21 in Arabidopsis (eudicot), with the highest number of 27 in poplar
155 Populus trichocarpa (eudicot). JmjC genes are also widespread in animals, from simple
156 invertebrates, such as sponge Amphimedon queenslandica, to mammals, such as human, with the
157 gene copy number ranging from 6 to 28, as well as in the unicellular M. brevicollis. Further
158 investigation reveals that the copy number variation in animals is mainly due to gene duplications
159 in vertebrates, also, the distribution of subgroup PKDM12 in different organisms is complex. In
160 fungi, there are fewer than 6 genes for each species, such as 1 in Schizosaccharomyces pombe, 3
161 in Saccharomyces cerevisiae and 5 in Agaricus bisporus var. bisporus, and altogether 27 JmjC
162 gene sequences were retrieved from 7 fungi.
163 In order to standardize gene name and be consistent with the literature, we adopted a
164 common nomenclature system based on the names of human genes according to the chromatin
165 modifying enzyme activities of animal members and Arabidopsis genes designated in previous
166 studies, as well as our phylogenetic analysis (Allis et al., 2007; Zhou and Ma, 2008). First, for
167 human and Arabidopsis genes with known functions or previous researches, the published gene
168 names were retained and used as a reference. Second, the orthologs of these genes from plants,
169 animals and fungi, respectively, were named following established reference. Ultimately, recent
170 paralogs were distinguished with an upper case letter after the number, using the same letter for
171 orthologs between organisms whenever possible.
172
173 Phylogenetic classification of JmjC genes into fourteen subfamilies
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174 To explore the evolutionary relationships of eukaryotic JmjC genes, we conducted
175 phylogenetic analyses with an alignment of the conserved JmjC domain from representative
176 species using NJ, ML and Bayesian methods. ML and Bayes analyses showed that proteins from
177 different species cluster together in clades with high support values, with support from NJ
178 analysis for most results. According to the results from phylogenetic and motif analyses, the
179 eukaryotic JmjC genes can be divided into fourteen subfamilies, designated as JARID2, JMJD6,
180 KDM2, KDM3, KDM4, KDM5, KDM6, PKDM7, PKDM8, PKDM9, PKDM10, PKDM11
181 PKDM12 and PKDM13 (Fig. 2). Among these subfamilies, KDM3, KDM5, JMJD6, PKDM11
182 and PKDM13 each contain members from plants, animals and fungi. On the other hand,
183 PKDM12 lacks fungal members and KDM2 and KDM4 lack plant members. In addition, PKDM7,
184 PKDM8 and PKDM9 are plant-specific groups, and JARID2, KDM6 and PKDM10 are animal
185 specific.
186 The fact that KDM3, KDM5, JMJD6, PKDM11 and PKDM13 subfamilies have members
187 from major multicellular groups of eukaryotes, including plants, animals and fungi, suggests that
188 these clades originated from five respective ancestral genes in the most recent common ancestor
189 (MRCA) of the three kingdoms. According to our phylogenetic analyses, JMJD6, PKDM11 and
190 PKDM13 cluster together with strong support, but some of the internal relationships of these
191 clades are not clear. The KDM2 and KDM4 subfamilies both contain animal and fungus JmjC
192 genes, indicating that the two clades are derived from ancestral genes that were present before the
193 separation of animals and fungi. The KDM4 clade is well supported by all phylogenetic methods,
194 but the classification of KDM2 also relied on the protein domain structures. The PKDM7,
195 PKDM8 and PKDM9 subfamilies are plant specific. According to the tree topology, the PKDM8
196 subfamily forms a sister group to PKDM9 and together they are from ancestral gene in the
197 MRCA of plants, animals and fungi, because they are sister to a large clade with genes from all
198 three kingdoms. Both PKDM7 and PKDM9 subfamilies contain genes from major lineages of
199 land plants, including Bryophyta, Pteridophyta and angiosperms, revealing that they are retained
200 in land plants. Whereas PKDM8 genes are conserved from Pteridophyta to angiosperms,
201 suggesting a likely loss of this subfamily in non-vascular plants. The JARID2, KDM6 and
202 PKDM10 subfamilies are composed of only animal genes. However, the sisterhood of JARID2
203 and KDM6 to other clades containing genes from all three kingdoms suggests that the origins of
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204 these subfamilies in the MRCA of three kingdoms; similarly, the ancestral gene of PKDM10 was
205 probably already present in the MRCA of animals and fungi. The early origins of JARID2 and
206 PKDM10 were further supported by identification of their homologs in M. brevicollis.
207 In summary, eukaryotic JmjC genes form fourteen subfamilies. These subfamilies can be
208 grouped into four categories: 5 shared by all three major eukaryotic kingdoms; 1 shared by
209 animals and plants; 2 found in both animals and fungi, with the counterparts loss from plants; 3
210 plant specific, as well as 3 animal specific. It is likely that there were at least 12 ancestral JmjC
211 genes present in the MRCA of three major eukaryotic kingdoms, with subsequent duplication and
212 losses in specific kingdoms (Fig. 2C). Additionally, within most subfamilies, except for JARID2,
213 PKDM11 and PKDM13, there are two or more gene copies in plants or animals, suggesting
214 further functional divergence of JmjC genes.
215
216 Different domain architecture and conserved non-JmjC motifs in subfamilies
217 According to the phylogenetic tree shown in Fig. 2A, fourteen monophyletic subfamilies
218 were identified in the JmjC gene family. These fourteen subfamilies represent twelve different
219 domain architectures, as three subfamilies (PKDM11, PKDM12 and PKDM13) possess only the
220 JmjC domain. Among these, the plant-specific PKDM7, PKDM8 and PKDM9 subfamilies have
221 similar domain architectures but PKDM7 proteins possess extra FYRN and FYRC domains. To
222 determine whether motifs outside the JmjC and other known domain were conserved between
223 members of the same subfamily, we searched for motifs in our dataset of JmjC proteins. We
224 found 40 motifs with lengths of greater than 10 amino acids that are specific within subfamilies
225 (Supplemental Table S3; Supplemental Fig. S1). The subfamilies exhibited various combinations
226 of motifs, yet similar with a subfamily, except for KDM3 and JMJD6, which have different
227 conserved motifs in plant and animal proteins (Fig. 2B). None of the 40 conserved motifs
228 corresponded to known domains in the Pfam database. The highly conserved motifs shared by
229 members of subfamilies of the JmjC domain proteins further support the classification presented
230 here. The conservation of these additional domains in respective subfamilies implies that they
231 play important roles in functions of these subfamilies.
232
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