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Histone Modifications Facilitate the Coexpression of Bidirectional Promoters in Rice

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Histone Modifications Facilitate the Coexpression of Bidirectional Promoters in Rice Fang et al. BMC Genomics (2016) 17:768 DOI 10.1186/s12864-016-3125-0 RESEARCH ARTICLE Open Access Histone modifications facilitate the coexpression of bidirectional promoters in rice Yuan Fang1, Lei Wang1, Ximeng Wang1, Qi You2, Xiucai Pan1, Jin Xiao1, Xiu-e Wang1, Yufeng Wu1, Zhen Su2*† and Wenli Zhang1,3*† Abstract Background: Bidirectional gene pairs are highly abundant and mostly co-regulated in eukaryotic genomes. The structural features of bidirectional promoters (BDPs) have been well studied in yeast, humans and plants. However, the underlying mechanisms responsible for the coexpression of BDPs remain understudied, especially in plants. Results: Here, we characterized chromatin features associated with rice BDPs. Several unique chromatin features were present in rice BDPs but were missing from unidirectional promoters (UDPs), including overrepresented active histone marks, canonical nucleosomes and underrepresented H3K27me3. In particular, overrepresented active marks (H3K4ac, H4K12ac, H4K16ac, H3K4me2 and H3K36me3) were truly overrepresented in type I BDPs but not in the other two BDPs, based on a Kolmogorov-Smirnov test. Conclusions: Our analyses indicate that active marks (H3K4ac, H4K12ac, H4K16ac, H3K4me3, H3K9ac and H3K27ac) may coordinate with repressive marks (H3K27me3 and H3K9me1/3) to build a unique chromatin structure that favors the coregulation of bidirectional gene pairs. Thus, our findings help to enhance the understanding of unique epigenetic mechanisms that regulate bidirectional gene pairs and may improve the manipulation of gene pairs for crop bioengineering. Keywords: Bidirectional promoters,regulation of gene expression, Coexpression, histone marks, Nucleosome positioning, Oryza sativa Background and fewer TATA boxes than those of UDPs [4, 5, 8]. The Bidirectional promoters (BDPs) regulate the bidirectional presence of overrepresented motifs, such as GABPA and transcription of protein-coding gene pairs with head-to- YY1, has already been recognized as a characteristic of head orientation, which means that the transcription of human BDPs [9–11]. Compared to UDPs, human BDPs each gene occurs on a different DNA strand and in oppos- have more epigenetic marks and chromatin related fea- ite directions. These promoters have been well character- tures, including RNA PolII binding sites, acetylation at ized in the eukaryotic genomes of yeast [1, 2], Drosophila H3, H3K9 and H3K27 and methylation at H3K4me2/3 [3], humans [4, 5] and some plants [6, 7]. Investigations of [9, 12]. By contrast, H4 acetylation is underrepresented BDPs in yeast and humans have shown that BDPs possess in human BDPs [11]. The majority of bidirectional gene unique features compared to unidirectional promoters pair products function in the same cellular pathway, (UDPs). The sequences of BDPs have higher GC contents and their involvement has been implicated in diverse processes, including DNA repair, the cell cycle, house- keeping, various metabolic pathways and human dis- * Correspondence: [email protected]; [email protected] †Equal contributors eases [4, 10, 13–19]. Although the coexpression of 2State Key Laboratory of Plant Physiology and Biochemistry, CBS, China bidirectional gene pairs is common in eukaryotic ge- Agricultural University, Beijing 100193, China nomes [5, 20–23], the detailed underlying mechanisms 1State Key Laboratory for Crop Genetics and Germplasm Enhancement, Nanjing Agriculture University, Nanjing, Jiangsu 210095, China that regulate coexpression are not well characterized. Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fang et al. BMC Genomics (2016) 17:768 Page 2 of 13 Thus, uncovering the unique regulatory mechanisms We then calculated the GC contents and observed associated with BDPs will provide new insights for un- approximately 54 %, 50 % and 45 % GC contents in derstanding eukaryotic gene regulation, especially co- types I, II and III BDPs, respectively (Additional file 1: regulation. Table S1). The GC contents in BDPs was significantly Progress has been made in characterizing plant BDPs higher than from randomly selected UDPs (Fig. 1).This in Arabidopsis [6, 24, 25], rice [6], maize [7] and Populus result confirmed the presence of GC-enriched se- [6] due to the recent availability of whole plant genome quences in eukaryotic BDPs [5, 9, 24]. In addition, the sequences and transcriptome data. Similar to BDPs in TATA box content was analyzed using the PLACE data- yeast and humans, plant BDPs have higher GC contents base [33]; TATA boxes were found in approximately and fewer TATA boxes than UDPs [6, 20, 24, 26]. 18 %, 52 % and 82 % of type I, II and III BDPs, respect- Moreover, plant BDPs are involved in the regulation of ively (Additional file 1: Table S1). The ratio of genes important agricultural traits [27–31]. However, infor- containing TATA boxes in type I BDPs was 30 % less mation on the chromatin related features of plant BDPs compared to randomly selected type I UDPs (random I) is still lacking. (Additional file 1: Table S1). In general, our analysis In this study, we continued to perform a comprehen- showed that GC content is inversely related to BDP sive analysis of chromatin-based epigenetic features in size. By contrast, TATA content is positively associated rice BDPs. BDPs were classified into three types (I, II with BDP size. Our results are the first to demonstrate and III with sizes of 0–250 bp, 250–500 bp and 500– that many rice type I BDPs are GC-rich sequences lack- 1000 bp, respectively) as described previously [32]. The ing TATA boxes. After comparing the expression of BDP size was defined as the intergenic distance be- gene pairs among the three BDP types, we found that tween the transcription start sites (TSSs) of the corre- type I BDPs had the highest expression level; whereas sponding gene pairs. We observed that type I BDPs type III BDPs had the lowest expression level. In (BDPs I) showed the highest percentage and strongest addition, we observed that the expression of one of level of coexpression, which was in agreement with the the gene pairs was significantly higher (higher FPKM, highest level of coexpression from gene pairs with p < 0.01)than its counterpart (lower FPKM, p < 0.01) 200 bp separating their TSSs. We also found several (data not shown). This result indicated that the GC unique chromatin features present in rice BDPs that or TATA content may affect the expression level of are not found in UDPs, including the overrepresenta- the corresponding genes. tion of active histone marks, canonical nucleosomes and the underrepresentation of H3K27me3. Strikingly, we found that overrepresented H3K4ac,H4K12ac, H4K16ac, H3K9ac and H3K27ac marks may play a sig- nificant role in the regulation of coexpressed gene pairs, indicating that histone acetylation functions in the co-regulation of gene pairs. Thus, our findings help to enhance the understanding of a unique epigenetic mechanism used in the regulation of BDPs, which could be used to improve the manipulation of gene pairs in crop bioengineering. Results DNA sequence features of rice bidirectional promoters To comprehensively characterize the DNA sequence pro- files of the BDPs in rice, we first identified bidirectional gene pairs with head-to-head orientations using the updated version of the rice genome (The Institute for Genomic Research (TIGR), rice subsp.Japonica version Fig. 1 Comparison of GC contents between BDPs and randomly 7.0) as described previously [32], which contains a total of selected UDPs. Type I, II and III BDPs: bidirectional promoters with intergenic sizes ranging from 0 to 250 bp; from 250 to 500 bp 55,801 annotated genes. We identified a total of 290 type I and from 500 to 1000 bp, respectively. R I, RII and RIII: randomly BDPs, 294 type II BDPs (BDPs II) and 627 type III BDPs selected unidirectional promoters (UDPs) with sizes as 250 bp, (BDPs III), with TSS intergenic distances of 0–250 bp 500 bp and 1000 bp starting from upstream of TSS of the (BDPs I), 250–500 bp (BDPs II) and 500–1000 bp (BDPs downstream genes, respectively, were used as controls for type I,II III), respectively. Our results were similar to the previously and III BDPs, respectively. Statistical analysis was conducted with a two-sample K-S test reported number of rice BDPs [24]. Fang et al. BMC Genomics (2016) 17:768 Page 3 of 13 Overrepresented motifs in rice BDPs involve in stress such as ACGTABREMOTIFA2OSEM (ACGTGKC) [37], responses CACGTGMOTIF (CACGTG) [39], CAMTA1(CCGCGT) To determine the occurrence of conserved motifs within [40] and ABRERATCAL(MACGYGB) [41, 42] (Additional rice BDPs, which are potential binding sites for trans- file 5: Table S5) were overrepresented in the promoters factors involved in the regulation of bidirectional gene of both drought-inducible rice gene pairs and unidirec- expression, we first classified BDPs into constitutive and tional genes that were upregulated under drought stress tissue-specific categories according to the expression pro- (Additional file 3: Table S3), This result was consistent files of the bidirectional gene pairs in three rice tissues with prior reports of overrepresented motifs in humans under normal conditions (leaf, callus and root) (Additional and plant BDPs compared to UDPs [5, 9, 24]. The pres- file 2: Table S2). We then identified the presence of over- ence of tissue-specific overrepresented motifs may play represented motifs with p-value cut-off of 0.05 using the an important role in regulating plant development and PLACE and PlantCare databases [34].
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