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. 2014 Aug 29;2:606–615. doi: 10.1016/j.mgene.2014.05.003

Genome wide analysis of Arabidopsis thaliana reveals high frequency of AAAGN7CTTT motif

Rajesh Mehrotra a,b,⁎, Vishesh Jain a, Chandra Shekhar c, Sandhya Mehrotra a
PMCID: PMC4288566  PMID: 25606443

Abstract

Sequence specific elements in DNA regulate transcription by recruiting transcription factors. The Dof proteins are a large family of transcription factors that share a single highly conserved zinc finger. The core to which Dof proteins bind has a consensus AAAG or ACTTTA sequence. These motifs have been over represented in many promoters. We performed a genome wide analysis of AAAG repeat elements increasing the spacer length from 0 to 25. Similar analyses was done with AAAG-CTTT motifs. We report unusual high frequency of AAAGN7CTTT in Arabidopsis thaliana genome. We also conclude that there is a preference for A/G nucleotides in spacer sequence between two AAAG repeats.

Keywords: Arabidopsis thaliana, Cis element, Designer promoter, Dof, Genome

Introduction

Promoters frequently contain multiple functional regulatory elements (Wray et al., 2003). This has an inherent question. How do redundancy and the evolution of cis element multiplicity take place. Cis elements are non coding DNA sequences present upstream of a gene and is required for proper spatio-temporal expression of the gene present downstream of it. It contains binding sites for transcription factors. The Dof domain proteins are typical example of plant specific transcription factors (Riechmann et al., 2000, Yanagisawa and Sheen, 1998, Yanagisawa, 2002). Dof transcription factor binds to a core sequence AAAG as shown by Vicente-Carbajosa et al. (1997) in a pull down assay. Dof domain proteins have been shown to interact with another class of transcription factors (Zhang et al., 1995). We are very much interested in knowing how transcription factors select their target-like sequences which are scattered on the entire chromosome and how they function at this site. We have shown earlier that the minimal core sequences of the commonly occurring cis elements can enhance promoter expression, even when used out of their native contexts (Mehrotra and Mehrotra, 2010, Mehrotra and Panwar, 2009, Mehrotra et al., 2005, Sawant et al., 2005). Using ACGT core sequence we showed that probabilistic model is not followed when we look for the evolution of cis element multiplicity (Mehrotra et al., 2012, Mehrotra et al., 2013). In this study we searched for the multiplicity of AAAG core sequence in the genome of Arabidopsis thaliana and reported that AAAGn7 CTTT is a preferred sequence in the genome. This information will be useful for designer promoters where specific interactions could be directed.

Methodology

The objective was to find out the frequency of the recurring sequences. Sequences of chromosomes were downloaded form the NCBI website (www.ncbi.nlm.nih.gov) and converted to a single line sequence using Notepad++. An ANSI C code was generated and later a code in Python 2.6.5 was used to find the results. The code written is as follows:

Table 1.

Frequency of two AAAG motifs separated by all possible distances (till 25 bp), across the five chromosomes. ‘n’ represents the intervening distance between the motifs. The second column displays the value of ‘n’.

chr1 chr2 chr3 chr4 chr5 Total
0 3224 2171 2501 1934 2908 12,738
AAAGnAAAG 1 2951 1873 2282 1711 2499 11,316
2 3314 2088 2546 2302 3215 13,465
3 2635 1755 2112 1693 2390 10,585
4 2732 1751 2038 1594 2377 10,492
5 2577 1746 2076 1570 2256 10,225
6 2529 1792 2107 1541 2373 10,342
7 2407 1663 2278 1589 2278 10,215
8 2533 1644 2134 1548 2341 10,200
9 2201 1454 1720 1330 2026 8731
10 2148 1518 1737 1390 2067 8860
11 2308 1543 1719 1365 2073 9008
12 2169 1454 1763 1274 2021 8681
13 2194 1438 1671 1352 1939 8594
14 2497 1501 1909 1428 2080 9415
15 2172 1435 1738 1348 2022 8715
16 2556 1507 1789 1439 2142 9433
17 2482 1690 2028 1583 2331 10,114
18 2154 1459 1888 1345 1925 8771
19 2230 1476 1819 1378 1917 8820
20 2338 1553 1776 1418 2105 9190
21 2144 1430 1646 1296 1939 8455
22 2129 1308 1609 1267 1881 8194
23 2159 1467 1733 1435 2015 8809
24 2254 1443 1689 1549 1997 8932
25 2147 1504 1721 1400 1923 8695
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Results and discussions

AAAGn7CTTT sequence is highly preferred in A. thaliana genome

Dof proteins, which are typically composed of 200–400 amino acids, are defined as DNA-binding proteins that have a highly conserved Dof domain. The strong similarity among Dof DNA-binding domains suggested that all Dof proteins display similar DNA-binding specificity. Indeed, an AAAG sequence or its reversibly oriented sequence, CTTT, is always found in the binding sequences of individual Dof proteins (Chen et al., 1996, dePaolis et al., 1996, Kang and Singh, 2000, Mena et al., 1998, Plesch et al., 2001, Washio, 2001, Yanagisawa and Izui, 1993) except a pumpkin Dof protein (AOBP) that recognizes an AGTA motif (Kisu et al., 1998). In A. thaliana, two AAAGs separated by one neuclotide is a known binding site for the OBP-1 protein (Yanagisawa, 2002). Similarly clusters of AAAG sites have been shown to additively contribute to guard cell-specificity of AtMYB60 promoter in guard cells (Cominelli et al., 2011). With an intention to discover potential new DOF binding sites in A. thaliana, the frequency of two AAAG or CTTT motifs separated by an increasing distance was carried out.

The frequency of AAAGAAAG without any spacer has a maximum occurrence of 12,738 as shown in Table 1 and Fig. 1 As we increase the spacer length, the frequency of occurrences started decreasing. There was a slight increase in frequency for the spacer length 14–17. Statistical analyses (data not shown) indicated them to be non significant as the deviation was essentially within 10–15%. Similar trend was observed for (CTTTnCTTT) as shown in Table 2 and Fig. 2.

Fig. 1.

Fig. 1

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Frequency of two AAAG motifs separated by all possible distances till 25 bp across the five chromosomes of Arabidopsis thaliana.

Table 2.

Frequency of two CTTT motifs separated by all possible distances (till 25 bp), across the five chromosomes. ‘n’ represents the intervening distance between the motifs. The second column displays the value of ‘n’.

chr1 chr2 chr3 chr4 chr5 Total
CTTTnCTTT 0 3195 2086 2447 1896 2764 12,388
1 3192 1910 2262 1755 2627 11,746
2 3269 2168 2465 2139 3060 13,101
3 2648 1706 2150 1677 2497 10,678
4 2616 1688 2081 1547 2374 10,306
5 2582 1723 2078 1606 2452 10,441
6 2591 1740 2014 1612 2344 10,301
7 2402 1708 2183 1664 2292 10,249
8 2416 1729 2097 1576 2287 10,105
9 2286 1448 1836 1390 2053 9013
10 2260 1528 1698 1384 2108 8978
11 2326 1531 1770 1381 2212 9220
12 2231 1484 1683 1293 1939 8630
13 2143 1484 1683 1407 1896 8613
14 2360 1606 1837 1435 2136 9374
15 2493 1523 1656 1431 1978 9081
16 2227 1494 1829 1477 2327 9354
17 2402 1673 2043 1568 2320 10,006
18 2237 1482 1797 1318 1985 8819
19 2240 1444 1657 1353 2001 8695
20 2305 1555 1746 1402 2101 9109
21 2180 1459 1610 1402 2045 8696
22 2124 1401 1578 1278 1946 8327
23 2218 1527 1747 1361 2014 8867
24 2156 1428 1625 1321 1916 8446
25 2219 1457 1703 1373 1986 8738
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Fig. 2.

Fig. 2

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Frequency of two CTTT motifs separated by all possible distances till 25 bp, across the five chromosomes of Arabidopsis thaliana.

A very interesting observation was made when we looked for combination of AAAG and CTTT sequences. An unexpected high frequency was observed for AAAGn7CTTT. The frequency of occurrence was observed as 14,977 which is more than two times the predecessor whose frequency is 7177 as shown in Table 3 and Fig. 3. However, when we change the orientation to CTTTn7 AAAG this tendency was not observed as shown in Table 4. The other implication of this is that transcriptional factor binding is direction specific. Not all AAAG motifs in plant promoters are targets of the Dof domain proteins. However, since an AAAG and a CTTT motif separated by a distance of 7 bp is present in an exceptionally high frequency, we think it is highly likely that this sequence combination may have a functional significance yet to be discovered.

Table 3.

Frequency of a AAAG and a CTTT motif separated by all possible distances (till 25 bp), across the five chromosomes. ‘n’ represents the intervening distance between the motifs. The second column displays the value of ‘n’.

AAAGnCTTT chr1 chr2 chr3 chr4 chr5 Total
0 2379 1352 1437 1570 2320 9058
1 1504 910 1236 905 1384 5939
2 1187 792 921 736 1018 4654
3 1398 903 993 792 1205 5291
4 1199 842 992 957 1190 5180
5 1308 863 995 795 1221 5182
6 1853 1205 1396 1069 1654 7177
7 3827 2482 2854 2358 3456 14,977
8 1546 990 1201 922 1350 6009
9 1534 1026 1197 994 1405 6156
10 1674 1050 1183 968 1366 6241
11 1544 1006 1218 1083 1633 6484
12 1620 976 1201 977 1470 6244
13 1557 1033 1180 974 1358 6102
14 1660 1081 1245 1012 1385 6383
15 1687 1119 1309 1032 1479 6626
16 1664 1107 1335 1016 1575 6697
17 1715 1092 1251 1135 1871 7064
18 1685 1119 1508 970 1454 6736
19 1518 992 1189 1014 1388 6101
20 1649 1040 1231 925 1354 6199
21 1673 1111 1269 951 1412 6416
22 1635 1046 1298 955 1454 6388
23 1548 1066 1196 937 1485 6232
24 1631 1059 1243 969 1461 6363
25 1655 1081 1278 977 1521 6512
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Fig. 3.

Fig. 3

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Frequency of CTTT and AAAG motifs separated by all possible distances till 25 bp, across the five chromosomes of Arabidopsis thaliana.

Table 4.

Frequency of a CTTT and a AAAG motif separated by all possible spacer distances (till 25 bp), across the five chromosomes. ‘n’ represents the intervening distance between the motifs. The second column displays the value of ‘n’.

chr1 chr2 chr3 chr4 chr5 Total
CTTTnAAAG 0 871 587 623 505 749 3335
1 1206 878 1005 736 1162 4987
2 1356 915 1097 833 1191 5392
3 1289 866 1065 818 1076 5114
4 1341 917 1381 871 1318 5828
5 1354 861 1039 785 1212 5251
6 1552 1003 1107 884 1403 5949
7 1461 1021 1118 924 1402 5926
8 1442 939 1100 832 1279 5592
9 1659 1052 1235 975 1375 6296
10 1635 941 1223 885 1406 6090
11 1689 1144 1590 1082 1635 7140
12 1697 1131 1279 1149 1854 7110
13 1606 1054 1223 1003 1544 6430
14 1433 1004 1117 878 1414 5846
15 1553 1060 1191 853 1442 6099
16 1703 1154 1214 1056 1471 6598
17 1629 1037 1203 961 1450 6280
18 1595 1016 1199 1002 1525 6337
19 1680 1135 1271 976 1484 6546
20 1579 1067 1256 957 1451 6310
21 1545 1040 1239 929 1392 6145
22 1632 1124 1251 1011 1459 6477
23 1541 1096 1265 998 1356 6256
24 1619 1002 1106 969 1406 6102
25 1650 1120 1201 952 1414 6337
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A and G are preferred as flanking nucleotides

We were interested to know which residues predominate in the flanking of AAAG sequence. Such studies are very important because many studies indicate that flanking sequences are very important for binding specificity (Foster et al., 1994, Izawa et al., 1993). We changed one nucleotide at a time following AAAG. As shown in Table 5, A and G predominate as flanking residues although there is an exception when (AAAG)–(AAAG) is separated by one nucleotide where the frequency of G flanking is 1918 which is less than C which is 2057. In all other cases G dominates as a flanking sequence over C and T.

Table 5.

Frequency of flanking nucleotide between two AAAG motifs separated by an increasing sequence length across five chromosomes.

AAAG?_AAAG CHR1 chr2 chr3 chr4 chr5 Total
A 1480 962 1143 885 1264 5734
G 487 306 399 296 430 1918
C 551 330 413 286 477 2057
T 433 275 327 244 328 1607



AA 725 441 574 403 646 2789
AG 402 248 286 234 327 1479
AC 189 124 135 115 366 929
AT 196 118 178 123 173 788



AAA 286 170 189 156 250 1051
AAG 152 105 143 96 136 632
AAC 74 41 59 48 55 277
AAT 65 37 28 36 43 209



AAAA 187 98 109 81 141 616
AAAG 122 79 95 65 118 479
AAAC 52 35 43 23 31 184
AAAT 33 18 23 19 38 131



AAAAA 76 42 69 49 66 302
AAAAG 33 26 45 30 37 171
AAAAC 22 21 13 14 15 85
AAAAT 15 13 11 6 18 63



AAAAAA 55 37 45 28 43 208
AAAAAG 20 13 18 9 27 87
AAAAAC 4 6 5 6 14 35
AAAAAT 8 1 6 6 2 22



AGA 102 85 87 72 108 454
AGG 47 32 39 25 49 192
AGC 32 30 30 27 26 145
AGT 55 34 47 41 59 236
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Conclusions

The promoter region of many genes contain multiple binding sites for the same transcription factor. One possibility is that individuals with multiple, redundant binding sites have higher fitness. Cis regulatory element multiplicity has been correlated with several gene functionalities like Promoters containing multiple sites evolve more slowly. In this paper we focused on the multiplicity of AAAG sequence with varied spacer lengths and also in combination with CTTT sequence. We report that AAAGn7 CTTT is a preferred sequence in the genome of A. thaliana. This information will be useful for designer promoters where specific interactions could be directed.

Acknowledgment

This work is part of UGC funded project of Dr Sandhya Mehrotra and Dr Rajesh Mehrotra. The authors are grateful to the administration of Birla Institute of Technology and Sciences, Pilani, Rajasthan for providing logistic support.

Footnotes

Appendix A

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.mgene.2014.05.003.

Appendix A. Supplementary data

Supplementary material

mmc1.docx (14.7KB, docx)

Supplementary material

mmc2.xlsx (9.4KB, xlsx)

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Associated Data

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Supplementary Materials

Supplementary material

mmc1.docx (14.7KB, docx)

Supplementary material

mmc2.xlsx (9.4KB, xlsx)

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