Comparative Biochemistry and Physiology, Part D 11 (2014) 45–48
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part D journal homepage: www.elsevier.com/locate/cbpd
Higher primates, but not New World monkeys, have a duplicate set of enhancers flanking their apoC-I genes Donald L. Puppione ⁎ The Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA
a r t i c l e
i n f o
Article history: Received 12 June 2014 Received in revised form 1 August 2014 Accepted 5 August 2014 Available online 13 August 2014 Keywords: Pseudogene Enhancer duplication Primates
a b s t r a c t Previous studies have demonstrated that the apoC-I gene and its pseudogene on human chromosome 19 are flanked by a duplicate set of enhancers. Multienhancers, ME.1 and ME.2, are located upstream from the genes and the hepatic control region enhancers, HCR.1 and HCR.2, are located downstream. The duplication of the enhancers has been thought to have occurred when the apoC-I gene was duplicated during primate evolution. Currently, the only primate data are for the human enhancers. Examining the genome of other primates (great and lesser apes, Old and New World monkeys), it was possible to locate the duplicate set of enhancers in apes and Old World monkeys. However, only a single set was found in New World monkeys. These observations provide additional evidence that the apoC-I gene and the flanking enhancers underwent duplication after the divergence of Old and New World monkeys. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Apolipoprotein C-I (apoC-I), the smallest of the soluble apolipoproteins, is also one of the most positively charged proteins in the circulation. It associates with high density lipoproteins (HDL) and the triacylglycerol-rich lipoproteins (Scanu and Edelstein, 2008). ApoC-I is an inhibitor of lipoprotein binding to the low density lipoprotein (LDL) receptor, LDL receptor-related protein, and very low density lipoprotein (VLDL) receptor (Shachter, 2001). It appears to interfere directly with fatty acid uptake and it is also the major plasma inhibitor of cholesteryl ester transfer protein (CETP) (Shachter, 2001). Discovery of this latter function arose from the seminal studies conducted on cholesterol-fed baboons (McGill et al., 1986). To keep a balance on the distribution of cholesteryl esters among the lipoproteins, CETP transfers primarily polyunsaturated cholesteryl esters from HDL to the less dense lipoproteins. Otherwise, the less dense lipoproteins would become enriched in monoenoic and saturated cholesteryl esters, derived from the liver. Interestingly, certain baboons responded to a diet enriched in lard and cholesterol by developing a high concentration of large, less dense HDL, designated HDL1 (McGill et al., 1986). Further
Abbreviations: OWM, Old World monkeys; NWM, New World monkeys; apoC-IB, the basic form of apoC-I in apes and OWM; apoC-IA, the acidic form of apoC-I in apes and OWM; ME, multienhancer; HCR, hepatic control region; mya, million years ago. ⁎ Boyer Hall, Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.cbd.2014.08.001 1744-117X/© 2014 Elsevier Inc. All rights reserved.
studies revealed that a truncated form of baboon apoC-I, containing the first 39 amino acids, was inhibiting CTEP (Kushwaha et al., 1993). The human gene for apoC-I and a pseudogene are located in a 44 kb gene cluster on chromosome 19 between the genes for apoE and apoCIV (Shih et al., 2000). In the other great apes, there are two functioning genes in this intergenic region of their chromosome 19. This is also the case for lesser apes and Old World monkeys (OWM) (Puppione and Whitelegge, 2013). One of the genes encodes a basic protein, designated apoC-IB, and the other encodes an acidic protein, designated apoC-IA (Puppione et al., 2010; Puppione and Whitelegge, 2013). ApoC-IB and apoC-IA are orthologous to human apoC-I and the pseudogene, respectively (Puppione and Whitelegge, 2013). Currently, it is not known whether apoC-IA and apoC-IB have different metabolic functions. The two genes for apoC-I arose through a duplication process that occurred after the divergence of NWM from the human lineage (Puppione and Whitelegge, 2013). The gene encoding apoC-IA became a pseudogene sometime between the divergence of bonobos and chimpanzees from the human lineage and the appearance of the Denisovans (Puppione and Whitelegge, 2013). Pseudogenization resulted when the codon for the penultimate amino acid in the signal sequence was changed to a stop codon (Puppione and Whitelegge, 2013). Studies have also shown that a duplicate set of enhancers flanks both the apoC-I gene and the pseudogene on human chromosome 19 (Shih et al., 2000). These enhancers have been reported to be essential for the expression of the apolipoprotein genes in the cluster on chromosome 19 (Shih et al., 2000; Mak et al., 2002). Those 5′ to the genes were designated as multienhancers (ME) (Shih et al., 2000). Those 3′ to the genes were designated as hepatic control region (HCR) enhancers
46
D.L. Puppione / Comparative Biochemistry and Physiology, Part D 11 (2014) 45–48
NWM
45429129 on chromosome 19. The two human hepatic control enhancer sequences each contain 154 bases, and they are located between 45427599 and 45427752 (HCR.1) and between 45439194 and 45439347 (HCR.2).
apoC-1 ME
HCR
2.2. Sources of primate sequences
OWM & Great Apes apoC-1A
apoC-1B ME.1
HCR.2
HCR.1 ME.2
Humans apo
apoC-1 ME.1
HCR.1 ME.2
HCR.2
Fig. 1. Orientation of the flanking enhancers around primate apoC-I genes. Footnote for Fig. 1. The human pseudogene, apoΨ.
(Simonet et al., 1993) (See Fig. 1). The enhancers and the apoC-I genes were presumed to have been duplicated at the same time (Allan et al., 1997; Shih et al., 2000). Both enhancers as well as their duplicates contain response elements for nuclear receptors. Within ME, there is a site for LXR (Liver X receptor) and within HCR a site for FXR (Farnesoid X-activated receptor). LXR and FXR are activated by oxysterols and bile acids, respectively, and it is becoming increasingly clear that these nuclear receptors are essential, not only in regulating cholesterol and bile acid metabolism, but also in the metabolism of sterols, fatty acids and glucose (Calkin and Tontonoz, 2012). LXR induces the expression of apoC-I gene as well as the other apolipoprotein genes in the cluster. FXR regulates the apoC-II gene and also the apoE gene (Calkin and Tontonoz, 2012). In humans, the enhancers vary slightly, with ME.1 being 95% identical to ME.2 and HCR.1 being 91% identical to HCR.2 (Allan et al., 1997; Shih et al., 2000). If the enhancers and the apoC-I gene were duplicated after the divergence of NWM from the human lineage, then we would predict that OWM should have a duplicate set and NWM a single set. To see if this were true, the corresponding apolipoprotein gene clusters for various primates were examined. Using the four human enhancer sequences, duplicate sets of enhancers were located in both great apes and in OWM, but only a single set of enhancers was found in NWM. The coordinates of these enhancer sequences are reported here for three great apes, two gibbons, three OWM and four NWM. Data on these enhancers expand our knowledge for this gene cluster in primates; the only primate data currently available are for the human enhancers. 2. Materials and methods 2.1. Detection and alignment of sequences Using the University of California at Santa Cruz (UCSC) Genome Browser as well as searching the Nucleotide database of the National Center for Biotechnology Information, it was possible to locate sequences that aligned with the four human enhancer sequences in the majority of the primates. Human ME.1 sequence with 620 bases can be found between coordinates 45415955 and 45416574 and the sequence of ME.2 with one less base is between 45428511 and
In the case of the three great apes, chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orangutan (Pongo abelii), coordinates were located on the respective chromosome 19 using the UCSC browser. For the white-cheeked gibbon (Nomascus leucogenys), the enhancers were on chromosome 17. Sequences for Mentawaii or Kloss's gibbon (Hylobates klossii) were found in GenBank entry (AC146473.1). The OWM GenBank entries were: AC145523 for hamadryas baboon (Papio hamadryas) and AC148222.2 for mantled guereza (Colobus guereza). JH682906, a reverse complement, in the UCSC database was the source for the olive baboon (Papio anubis). The NWM GenBank entries were AC146285.3 for dusky or red-bellied titi (Callicebus moloch), AC14620.2 for Ma's night monkey (Aotus nancymaae), and AC151887.2 for squirrel monkey (Saimiri boliviensis). The marmoset (Callithrix jacchus) sequences were located on chromosome 22 using the UCSC browser. 3. Results 3.1. Primate apoC-I genes Primate apoC-I genes have four exons and they encode proteins with molecular weights around 7 kD. As the coordinates listed in Tables 1 and 2 indicate, the genes range in size from 3 kb in NWM to over 6 kb in lesser apes. The differences in size are primarily due to the insertion of Alu sequences during the course of primate evolution. In most cases the gene coordinates shown in the Tables are from the beginning of exon 2 to the end of exon 4. Exon 1 of the apoC-I gene is non-coding. Several of the genes are incomplete due to sequence gaps. The gorilla apoC-IB gene I is missing exon 3. Both exons 3 and 4 are missing from the Nomascus apoC-IB gene and exon 4 is missing from the Hylobates apoC-IA gene. Exon 3 is missing from apoC-IA genes of both baboons. 3.2. Primate multienhancers The multienhancer sequences of the ape and OWM were very similar to the human sequences. All had between 610 and 620 bases, except Nomascus ME.2 with 599. The single multienhancers in the NWM were larger, averaging around 656 bases. Alignment of NMW sequences with the smaller human enhancer sequences revealed that the size difference was due to the deletion of 39 nucleotides having this sequence, AGGC AGGAGC TTTGCA G/A TTC TATTCTTGTG GGGTCAGGA. At the site indicated in italics, three of the NWM sequences had a guanine whereas the Dusky titi sequence had an adenine. As Fig. 1 indicates, NWM have a single multienhancer and a single HCR. The previous studies had shown that the nuclear receptor LXR interacts with a 24-base response element present in both human multienhancers (Shih et al., 2000). In both cases, the sequence was GCCAGGGTCACTGGCGGTCAAAGG. Identical sequences were found in multienhancers of the primates listed in Tables 1 and 2, with two exceptions. Gorilla ME2 had a single change with a thymine instead of a cytosine at the second position and Ma's night monkey's single enhancer ended with two changes, GAAG instead of AAGG. In all the multienhancers listed in Tables 1 and 2, the sequence could be found three-quarters the way down from the start. 3.3. Primate hepatic control region Due to sequence gaps the complete sequences for both great ape HCR were not located. In the case of HCR.1, only the gorilla had a complete sequence, but a partial sequence lacking the first 40 bases was
D.L. Puppione / Comparative Biochemistry and Physiology, Part D 11 (2014) 45–48
47
Table 1 Coordinates of flanking enhancers and apoC-I genes in apes and Old World monkeys. Great apes
Chimpanzee
Gorilla
Orangutan
ME.1 apoC-IB gene HCR.1 ME.2 apoC-IA gene HCR.2
50102840–50103458 50105028–50109366 Sequence gap 50114761–50115379 50116491–50120477 50125732–50125885
42122414–42123033 42124612–42129314 42133480–42133633 42134394–42135011 42137059–42141439 Sequence gap
46153834–46154445 46156018–46160253 Sequence gap 46166897–46167516 46168619–46173056 46180047–46180200
Lesser apes
Nomascus l..
Hylobates k.
ME.1 apoC-IB gene HCR1. ME.2 apoC-IA gene HCR.2
91239273–91239892 91241477–91241534 91249384–91249537 91250286–91250884 Sequence gap 91257799–9125792
18511–19124 20697–26762 31851–32004 32764–33382 34554–35440 Sequence gap
OWM
Papio a.
Papio h.
Colobus
ME.1 apoC-IB gene HCR.1 ME.2 apoC-IA gene HCR.2
89414–90032 84609–88330 80174–80327 78146–78555 73815–77042 68499–68652
143649–144267 145351–149539 153724–153877 155409–156018 157122–161163 166013–166166
166310–166929 168001–172552 176970–177123 178327–178939 180044–185034 189586–189739
located for orangutan HCR.1 between 46152964 and 46153077. The gorilla HCR.2 was not located (Table 1). HCR.1 was found for both gibbons, but HCR.2 was detected in only Nomascus entry. Both hepatic control regions were found in each of the entries for the three OWM (Table 1). A single HCR was found in each of the four NWM (Table 2). FXR response elements are located toward the terminal region of both human hepatic control regions, having the identical sequence AGGTCAGAGACCT (Kast et al., 2001). This was also true for the other primates in which the same sequence was found with four exceptions. These differences, indicated in italics, were AGGTCAGAGACGT in gorilla HCR.1 and AGGTTAGAGACCT in the orangutan HCR.2. For the gibbon HCR.1, the sequences were AGGTCGGAGACCT (Nomascus) and AGGT CAGGGACCT (Hylobates). The 13-nucleotide sequence of New World monkeys was identical to the human sequence. 4. Discussion Based on comparative analyses of the rate of non-synonymous amino acid substitution, it was concluded that apoC-I was evolving more rapidly than any of the other soluble apolipoproteins (Luo et al., 1989). During primate evolution, the apoC-I gene was duplicated and subsequently one of the duplicates was converted to a pseudogene. Estimates of the time of duplication have varied between 35 and 40 mya (Luo et al., 1989; Raisonnier, 1991; Freitas et al., 2000). Comparing synonymous and non-synonymous changes in the human pseudogene and its functional counterpart, Luo et al. concluded that the pseudogenization probably occurred prior to the divergence of New World monkeys from the human lineage when apoC-I was duplicated (Luo et al., 1989). These authors also suggested that the pseudogene might also be detected in New World monkeys. This has
turned out not to be the case. The examination of the genomic data for five New World monkeys revealed only a single apoC-I gene (Puppione and Whitelegge, 2013). Moreover, for the four NWM reported in this study, the sequences between HCR and apoC-IV did not contain a duplicate set of enhancers. Although not analyzed in this current study, the prosimian bushbaby (Otolemur garnettii) also has a single gene for apoC-I (Puppione and Whitelegge, 2013). The hepatic control region was the first of the two enhancers to be reported and it was shown to be essential in the expression of both apoE and apoC-I (Simonet et al., 1993). Initially it was thought to be localized to a 764-base pair region, but it was found later to consist of just 154 base pairs downstream from the apoC-I gene (Shachter et al., 1993). About the same time, the second hepatic control region was identified downstream of the human pseudogene (Allan et al., 1995). The response element for FXR was identified in both human HCR and is located toward the terminus. The multienhancer region, the second set of enhancers to be identified, was later found as duplicates as well, with ME.1 upstream from the apoC-I gene and ME.2 upstream from the pseudogene (Shih et al., 2000) (See Fig. 1). Both these enhancers contain the functional response element for LXR. With the observation of two set enhancers flanking the apoC-I gene and the pseudogene on human chromosome 19, it was concluded that the enhancers were duplicated at the time of apoC-I duplication (Allan et al., 1995; Shih et al., 2000). This current examination of primate genomic data is consistent with that conclusion. Duplicate sets of the multienhancer and the hepatic control region were found in apes and Old World monkeys but not in New World monkeys. It also provides additional evidence that the duplication took place after, but not before, the divergence of NWM from the human lineage.
Table 2 Coordinates of flanking enhancers and apoC-I genes in NWM. NWM
Dusky titi
Marmoset
Ma's monkey
Sq monkey
ME apoC-I gene HCR
138994–139652 141287–143979 149316–149473
36963853–36964509 36965986–36970402 36974875–36975032
45084–45736 47233–49965 52918–53075
157076–157733 159235–162244 166491–166648
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The primate enhancers located in this study have been reported to enable both FXR and LXR to regulate key genes in lipoprotein metabolism. Using macrophage cells, LXR was demonstrated to regulate the expression of the four apolipoprotein genes in the clusters on human chromosome 19 and mouse chromosome 7 (Mak et al., 2002). If similar studies were done with macrophages obtained from either apes or Old World monkeys, it would be interesting to see if there would be a difference in the expression of apoC-IA and apoC-IB genes. Currently it has been estimated that there are over 14,000 pseudogenes in the human genome (Pei et al., 2012). Many of them, like the gene for apoC-IA, most likely are functionally active in other primates. If these nonhuman genes were identified, the metabolism of the resulting proteins could potentially be studied in the future. These studies could very well provide new insight into the evolution and physiology of humans. Acknowledgments The author is grateful to Dr. Jerzy Jurka and Dr. Kenji Kojima of the Genetic Information Research Institute, Mountain View, CA for providing genomic data for the New World monkeys and to Dr. Peter Edwards of UCLA's Department of Biological Chemistry for his comments and suggestions. References Allan, C.M., Walker, D., Taylor, J.M., 1995. Evolutionary duplication of a hepatic control region in the human apolipoprotein E gene locus. Identification of a second region that confers high level and liver-specific expression of the human apolipoprotein E gene in transgenic mice. J. Biol. Chem. 270, 26278–26281. Allan, C.M., Taylor, S., Taylor, J.M., 1997. Two hepatic enhancers, HCR.1 and HCR.2, coordinate the liver expression of the entire human apolipoprotein E/C-I/C-IV/C-II gene cluster. J. Biol. Chem. 272, 29113–29119. Calkin, A.C., Tontonoz, P., 2012. Transcriptional integration of metabolism by the nuclear sterol activated receptors LXR and FXR. Nat. Rev. Mol. Cell Biol. 13, 213–224. Freitas, E.M., Gaudieri, S., Zhang, W.J., Kulski, J.K., van Bockxmeer, F.M., Christiansen, F.T., Dawkins, R.L., 2000. Duplication and diversification of the apolipoprotein CI
(APOCI) genomic segment in association with retroelements. J. Mol. Evol. 50, 391–396. Kast, H.R., Nguyen, C.M., Sinal, C.J., Jones, S.A., Laffitte, B.A., Reue, K., Gonzalez, F.J., Willson, T.M., Edwards, P.A., 2001. Farnesoid X-activated receptor induces apolipoprotein C-II transcription: a molecular mechanism linking plasma triglyceride levels to bile acids. Mol. Endocrinol. 15, 1720–1728. Kushwaha, R.S., Hasan, S.Q., McGill Jr., H.C., Getz, G.S., Dunham, R.G., Kanda, P., 1993. Characterization of cholesteryl ester transfer protein inhibitor from plasma of baboons (Papio sp.). J. Lipid Res. 34, 1285–1297. Luo, C.C., Li, W.H., Chan, L., 1989. Structure and expression of dog apolipoprotein A-I, E, and C-I mRNAs: implications for the evolution and functional constraints of apolipoprotein structure. J. Lipid Res. 30, 1735–1746. Mak, P.A., Laffitte, B.A., Desrumaux, C., Joseph, S.B., Curtiss, L.K., Mangelsdorf, D.J., Tontonoz, P., Edwards, P.A., 2002. Regulated expression of the apolipoprotein E/C-I/ C-IV/C-II gene cluster in murine and human macrophages. A critical role for nuclear liver X receptors alpha and beta. J. Biol. Chem. 277, 31900–31908. McGill Jr., H.C., McMahan, C.A., Kushwiha, K.S., Mott, G.E., Carey, K.D., 1986. Dietary effects on serum lipoproteins of dyslipoproteinemic baboons with high HDLI. Arteriosclerosis 6, 651–663. Pei, B., Sisu, C., Frankish, A., Howald, C., Habegger, L., Mu, X.J., Harte, R., Balasubramanian, S., Tanzer, A., Diekhans, M., Reymond, A., Hubbard, T., Harrow, J., Gerstein, M.B., 2012. The GENCODE pseudogene resource. Genome Biol. 13, R51. Puppione, D.L., Whitelegge, J.P., 2013. Proteogenomic review of the changes in primate apoC-I during evolution. Front. Biol. 8, 533–548. Puppione, D.L., Ryan, C.M., Bassilian, S., Souda, P., Xiao, X., Ryder, O.A., Whitelegge, J.P., 2010. Detection of two distinct forms of apoC-I in great apes. Comp. Biochem. Physiol. D Genomics Proteomics 7, 9–13. Raisonnier, A., 1991. Duplication of the apolipoprotein C-I gene occurred about forty million years ago. J. Mol. Evol. 32, 211–219. Scanu, A.M., Edelstein, C., 2008. HDL: bridging past and present with a look at the future. FASEB J. 22, 4044–4054. Shachter, N.S., 2001. Apolipoproteins C-I and C-III as important modulators of lipoprotein metabolism. Curr. Opin. Lipidol. 12, 297–304. Shachter, N.S., Zhu, Y., Walsh, A., Breslow, J.L., Smith, J.D., 1993. Localization of a liverspecific enhancer in the apolipoprotein E/C-I/C-II gene locus. J. Lipid Res. 34, 1699–1707. Shih, S.J., Allan, C., Grehan, S., Tse, E., Moran, C., Taylor, J.M., 2000. Duplicated downstream enhancers control expression of the human apolipoprotein E gene in macrophages and adipose tissue. J. Biol. Chem. 275, 31567–31572. Simonet, W.S., Bucay, N., Lauer, S.J., Taylor, J.M., 1993. A far-downstream hepatocytespecific control region directs expression of the linked human apolipoprotein E and C-I genes in transgenic mice. J. Biol. Chem. 268, 8221–8229.
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part D journal homepage: www.elsevier.com/locate/cbpd
Higher primates, but not New World monkeys, have a duplicate set of enhancers flanking their apoC-I genes Donald L. Puppione ⁎ The Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA
a r t i c l e
i n f o
Article history: Received 12 June 2014 Received in revised form 1 August 2014 Accepted 5 August 2014 Available online 13 August 2014 Keywords: Pseudogene Enhancer duplication Primates
a b s t r a c t Previous studies have demonstrated that the apoC-I gene and its pseudogene on human chromosome 19 are flanked by a duplicate set of enhancers. Multienhancers, ME.1 and ME.2, are located upstream from the genes and the hepatic control region enhancers, HCR.1 and HCR.2, are located downstream. The duplication of the enhancers has been thought to have occurred when the apoC-I gene was duplicated during primate evolution. Currently, the only primate data are for the human enhancers. Examining the genome of other primates (great and lesser apes, Old and New World monkeys), it was possible to locate the duplicate set of enhancers in apes and Old World monkeys. However, only a single set was found in New World monkeys. These observations provide additional evidence that the apoC-I gene and the flanking enhancers underwent duplication after the divergence of Old and New World monkeys. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Apolipoprotein C-I (apoC-I), the smallest of the soluble apolipoproteins, is also one of the most positively charged proteins in the circulation. It associates with high density lipoproteins (HDL) and the triacylglycerol-rich lipoproteins (Scanu and Edelstein, 2008). ApoC-I is an inhibitor of lipoprotein binding to the low density lipoprotein (LDL) receptor, LDL receptor-related protein, and very low density lipoprotein (VLDL) receptor (Shachter, 2001). It appears to interfere directly with fatty acid uptake and it is also the major plasma inhibitor of cholesteryl ester transfer protein (CETP) (Shachter, 2001). Discovery of this latter function arose from the seminal studies conducted on cholesterol-fed baboons (McGill et al., 1986). To keep a balance on the distribution of cholesteryl esters among the lipoproteins, CETP transfers primarily polyunsaturated cholesteryl esters from HDL to the less dense lipoproteins. Otherwise, the less dense lipoproteins would become enriched in monoenoic and saturated cholesteryl esters, derived from the liver. Interestingly, certain baboons responded to a diet enriched in lard and cholesterol by developing a high concentration of large, less dense HDL, designated HDL1 (McGill et al., 1986). Further
Abbreviations: OWM, Old World monkeys; NWM, New World monkeys; apoC-IB, the basic form of apoC-I in apes and OWM; apoC-IA, the acidic form of apoC-I in apes and OWM; ME, multienhancer; HCR, hepatic control region; mya, million years ago. ⁎ Boyer Hall, Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.cbd.2014.08.001 1744-117X/© 2014 Elsevier Inc. All rights reserved.
studies revealed that a truncated form of baboon apoC-I, containing the first 39 amino acids, was inhibiting CTEP (Kushwaha et al., 1993). The human gene for apoC-I and a pseudogene are located in a 44 kb gene cluster on chromosome 19 between the genes for apoE and apoCIV (Shih et al., 2000). In the other great apes, there are two functioning genes in this intergenic region of their chromosome 19. This is also the case for lesser apes and Old World monkeys (OWM) (Puppione and Whitelegge, 2013). One of the genes encodes a basic protein, designated apoC-IB, and the other encodes an acidic protein, designated apoC-IA (Puppione et al., 2010; Puppione and Whitelegge, 2013). ApoC-IB and apoC-IA are orthologous to human apoC-I and the pseudogene, respectively (Puppione and Whitelegge, 2013). Currently, it is not known whether apoC-IA and apoC-IB have different metabolic functions. The two genes for apoC-I arose through a duplication process that occurred after the divergence of NWM from the human lineage (Puppione and Whitelegge, 2013). The gene encoding apoC-IA became a pseudogene sometime between the divergence of bonobos and chimpanzees from the human lineage and the appearance of the Denisovans (Puppione and Whitelegge, 2013). Pseudogenization resulted when the codon for the penultimate amino acid in the signal sequence was changed to a stop codon (Puppione and Whitelegge, 2013). Studies have also shown that a duplicate set of enhancers flanks both the apoC-I gene and the pseudogene on human chromosome 19 (Shih et al., 2000). These enhancers have been reported to be essential for the expression of the apolipoprotein genes in the cluster on chromosome 19 (Shih et al., 2000; Mak et al., 2002). Those 5′ to the genes were designated as multienhancers (ME) (Shih et al., 2000). Those 3′ to the genes were designated as hepatic control region (HCR) enhancers
46
D.L. Puppione / Comparative Biochemistry and Physiology, Part D 11 (2014) 45–48
NWM
45429129 on chromosome 19. The two human hepatic control enhancer sequences each contain 154 bases, and they are located between 45427599 and 45427752 (HCR.1) and between 45439194 and 45439347 (HCR.2).
apoC-1 ME
HCR
2.2. Sources of primate sequences
OWM & Great Apes apoC-1A
apoC-1B ME.1
HCR.2
HCR.1 ME.2
Humans apo
apoC-1 ME.1
HCR.1 ME.2
HCR.2
Fig. 1. Orientation of the flanking enhancers around primate apoC-I genes. Footnote for Fig. 1. The human pseudogene, apoΨ.
(Simonet et al., 1993) (See Fig. 1). The enhancers and the apoC-I genes were presumed to have been duplicated at the same time (Allan et al., 1997; Shih et al., 2000). Both enhancers as well as their duplicates contain response elements for nuclear receptors. Within ME, there is a site for LXR (Liver X receptor) and within HCR a site for FXR (Farnesoid X-activated receptor). LXR and FXR are activated by oxysterols and bile acids, respectively, and it is becoming increasingly clear that these nuclear receptors are essential, not only in regulating cholesterol and bile acid metabolism, but also in the metabolism of sterols, fatty acids and glucose (Calkin and Tontonoz, 2012). LXR induces the expression of apoC-I gene as well as the other apolipoprotein genes in the cluster. FXR regulates the apoC-II gene and also the apoE gene (Calkin and Tontonoz, 2012). In humans, the enhancers vary slightly, with ME.1 being 95% identical to ME.2 and HCR.1 being 91% identical to HCR.2 (Allan et al., 1997; Shih et al., 2000). If the enhancers and the apoC-I gene were duplicated after the divergence of NWM from the human lineage, then we would predict that OWM should have a duplicate set and NWM a single set. To see if this were true, the corresponding apolipoprotein gene clusters for various primates were examined. Using the four human enhancer sequences, duplicate sets of enhancers were located in both great apes and in OWM, but only a single set of enhancers was found in NWM. The coordinates of these enhancer sequences are reported here for three great apes, two gibbons, three OWM and four NWM. Data on these enhancers expand our knowledge for this gene cluster in primates; the only primate data currently available are for the human enhancers. 2. Materials and methods 2.1. Detection and alignment of sequences Using the University of California at Santa Cruz (UCSC) Genome Browser as well as searching the Nucleotide database of the National Center for Biotechnology Information, it was possible to locate sequences that aligned with the four human enhancer sequences in the majority of the primates. Human ME.1 sequence with 620 bases can be found between coordinates 45415955 and 45416574 and the sequence of ME.2 with one less base is between 45428511 and
In the case of the three great apes, chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orangutan (Pongo abelii), coordinates were located on the respective chromosome 19 using the UCSC browser. For the white-cheeked gibbon (Nomascus leucogenys), the enhancers were on chromosome 17. Sequences for Mentawaii or Kloss's gibbon (Hylobates klossii) were found in GenBank entry (AC146473.1). The OWM GenBank entries were: AC145523 for hamadryas baboon (Papio hamadryas) and AC148222.2 for mantled guereza (Colobus guereza). JH682906, a reverse complement, in the UCSC database was the source for the olive baboon (Papio anubis). The NWM GenBank entries were AC146285.3 for dusky or red-bellied titi (Callicebus moloch), AC14620.2 for Ma's night monkey (Aotus nancymaae), and AC151887.2 for squirrel monkey (Saimiri boliviensis). The marmoset (Callithrix jacchus) sequences were located on chromosome 22 using the UCSC browser. 3. Results 3.1. Primate apoC-I genes Primate apoC-I genes have four exons and they encode proteins with molecular weights around 7 kD. As the coordinates listed in Tables 1 and 2 indicate, the genes range in size from 3 kb in NWM to over 6 kb in lesser apes. The differences in size are primarily due to the insertion of Alu sequences during the course of primate evolution. In most cases the gene coordinates shown in the Tables are from the beginning of exon 2 to the end of exon 4. Exon 1 of the apoC-I gene is non-coding. Several of the genes are incomplete due to sequence gaps. The gorilla apoC-IB gene I is missing exon 3. Both exons 3 and 4 are missing from the Nomascus apoC-IB gene and exon 4 is missing from the Hylobates apoC-IA gene. Exon 3 is missing from apoC-IA genes of both baboons. 3.2. Primate multienhancers The multienhancer sequences of the ape and OWM were very similar to the human sequences. All had between 610 and 620 bases, except Nomascus ME.2 with 599. The single multienhancers in the NWM were larger, averaging around 656 bases. Alignment of NMW sequences with the smaller human enhancer sequences revealed that the size difference was due to the deletion of 39 nucleotides having this sequence, AGGC AGGAGC TTTGCA G/A TTC TATTCTTGTG GGGTCAGGA. At the site indicated in italics, three of the NWM sequences had a guanine whereas the Dusky titi sequence had an adenine. As Fig. 1 indicates, NWM have a single multienhancer and a single HCR. The previous studies had shown that the nuclear receptor LXR interacts with a 24-base response element present in both human multienhancers (Shih et al., 2000). In both cases, the sequence was GCCAGGGTCACTGGCGGTCAAAGG. Identical sequences were found in multienhancers of the primates listed in Tables 1 and 2, with two exceptions. Gorilla ME2 had a single change with a thymine instead of a cytosine at the second position and Ma's night monkey's single enhancer ended with two changes, GAAG instead of AAGG. In all the multienhancers listed in Tables 1 and 2, the sequence could be found three-quarters the way down from the start. 3.3. Primate hepatic control region Due to sequence gaps the complete sequences for both great ape HCR were not located. In the case of HCR.1, only the gorilla had a complete sequence, but a partial sequence lacking the first 40 bases was
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Table 1 Coordinates of flanking enhancers and apoC-I genes in apes and Old World monkeys. Great apes
Chimpanzee
Gorilla
Orangutan
ME.1 apoC-IB gene HCR.1 ME.2 apoC-IA gene HCR.2
50102840–50103458 50105028–50109366 Sequence gap 50114761–50115379 50116491–50120477 50125732–50125885
42122414–42123033 42124612–42129314 42133480–42133633 42134394–42135011 42137059–42141439 Sequence gap
46153834–46154445 46156018–46160253 Sequence gap 46166897–46167516 46168619–46173056 46180047–46180200
Lesser apes
Nomascus l..
Hylobates k.
ME.1 apoC-IB gene HCR1. ME.2 apoC-IA gene HCR.2
91239273–91239892 91241477–91241534 91249384–91249537 91250286–91250884 Sequence gap 91257799–9125792
18511–19124 20697–26762 31851–32004 32764–33382 34554–35440 Sequence gap
OWM
Papio a.
Papio h.
Colobus
ME.1 apoC-IB gene HCR.1 ME.2 apoC-IA gene HCR.2
89414–90032 84609–88330 80174–80327 78146–78555 73815–77042 68499–68652
143649–144267 145351–149539 153724–153877 155409–156018 157122–161163 166013–166166
166310–166929 168001–172552 176970–177123 178327–178939 180044–185034 189586–189739
located for orangutan HCR.1 between 46152964 and 46153077. The gorilla HCR.2 was not located (Table 1). HCR.1 was found for both gibbons, but HCR.2 was detected in only Nomascus entry. Both hepatic control regions were found in each of the entries for the three OWM (Table 1). A single HCR was found in each of the four NWM (Table 2). FXR response elements are located toward the terminal region of both human hepatic control regions, having the identical sequence AGGTCAGAGACCT (Kast et al., 2001). This was also true for the other primates in which the same sequence was found with four exceptions. These differences, indicated in italics, were AGGTCAGAGACGT in gorilla HCR.1 and AGGTTAGAGACCT in the orangutan HCR.2. For the gibbon HCR.1, the sequences were AGGTCGGAGACCT (Nomascus) and AGGT CAGGGACCT (Hylobates). The 13-nucleotide sequence of New World monkeys was identical to the human sequence. 4. Discussion Based on comparative analyses of the rate of non-synonymous amino acid substitution, it was concluded that apoC-I was evolving more rapidly than any of the other soluble apolipoproteins (Luo et al., 1989). During primate evolution, the apoC-I gene was duplicated and subsequently one of the duplicates was converted to a pseudogene. Estimates of the time of duplication have varied between 35 and 40 mya (Luo et al., 1989; Raisonnier, 1991; Freitas et al., 2000). Comparing synonymous and non-synonymous changes in the human pseudogene and its functional counterpart, Luo et al. concluded that the pseudogenization probably occurred prior to the divergence of New World monkeys from the human lineage when apoC-I was duplicated (Luo et al., 1989). These authors also suggested that the pseudogene might also be detected in New World monkeys. This has
turned out not to be the case. The examination of the genomic data for five New World monkeys revealed only a single apoC-I gene (Puppione and Whitelegge, 2013). Moreover, for the four NWM reported in this study, the sequences between HCR and apoC-IV did not contain a duplicate set of enhancers. Although not analyzed in this current study, the prosimian bushbaby (Otolemur garnettii) also has a single gene for apoC-I (Puppione and Whitelegge, 2013). The hepatic control region was the first of the two enhancers to be reported and it was shown to be essential in the expression of both apoE and apoC-I (Simonet et al., 1993). Initially it was thought to be localized to a 764-base pair region, but it was found later to consist of just 154 base pairs downstream from the apoC-I gene (Shachter et al., 1993). About the same time, the second hepatic control region was identified downstream of the human pseudogene (Allan et al., 1995). The response element for FXR was identified in both human HCR and is located toward the terminus. The multienhancer region, the second set of enhancers to be identified, was later found as duplicates as well, with ME.1 upstream from the apoC-I gene and ME.2 upstream from the pseudogene (Shih et al., 2000) (See Fig. 1). Both these enhancers contain the functional response element for LXR. With the observation of two set enhancers flanking the apoC-I gene and the pseudogene on human chromosome 19, it was concluded that the enhancers were duplicated at the time of apoC-I duplication (Allan et al., 1995; Shih et al., 2000). This current examination of primate genomic data is consistent with that conclusion. Duplicate sets of the multienhancer and the hepatic control region were found in apes and Old World monkeys but not in New World monkeys. It also provides additional evidence that the duplication took place after, but not before, the divergence of NWM from the human lineage.
Table 2 Coordinates of flanking enhancers and apoC-I genes in NWM. NWM
Dusky titi
Marmoset
Ma's monkey
Sq monkey
ME apoC-I gene HCR
138994–139652 141287–143979 149316–149473
36963853–36964509 36965986–36970402 36974875–36975032
45084–45736 47233–49965 52918–53075
157076–157733 159235–162244 166491–166648
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The primate enhancers located in this study have been reported to enable both FXR and LXR to regulate key genes in lipoprotein metabolism. Using macrophage cells, LXR was demonstrated to regulate the expression of the four apolipoprotein genes in the clusters on human chromosome 19 and mouse chromosome 7 (Mak et al., 2002). If similar studies were done with macrophages obtained from either apes or Old World monkeys, it would be interesting to see if there would be a difference in the expression of apoC-IA and apoC-IB genes. Currently it has been estimated that there are over 14,000 pseudogenes in the human genome (Pei et al., 2012). Many of them, like the gene for apoC-IA, most likely are functionally active in other primates. If these nonhuman genes were identified, the metabolism of the resulting proteins could potentially be studied in the future. These studies could very well provide new insight into the evolution and physiology of humans. Acknowledgments The author is grateful to Dr. Jerzy Jurka and Dr. Kenji Kojima of the Genetic Information Research Institute, Mountain View, CA for providing genomic data for the New World monkeys and to Dr. Peter Edwards of UCLA's Department of Biological Chemistry for his comments and suggestions. References Allan, C.M., Walker, D., Taylor, J.M., 1995. Evolutionary duplication of a hepatic control region in the human apolipoprotein E gene locus. Identification of a second region that confers high level and liver-specific expression of the human apolipoprotein E gene in transgenic mice. J. Biol. Chem. 270, 26278–26281. Allan, C.M., Taylor, S., Taylor, J.M., 1997. Two hepatic enhancers, HCR.1 and HCR.2, coordinate the liver expression of the entire human apolipoprotein E/C-I/C-IV/C-II gene cluster. J. Biol. Chem. 272, 29113–29119. Calkin, A.C., Tontonoz, P., 2012. Transcriptional integration of metabolism by the nuclear sterol activated receptors LXR and FXR. Nat. Rev. Mol. Cell Biol. 13, 213–224. Freitas, E.M., Gaudieri, S., Zhang, W.J., Kulski, J.K., van Bockxmeer, F.M., Christiansen, F.T., Dawkins, R.L., 2000. Duplication and diversification of the apolipoprotein CI
(APOCI) genomic segment in association with retroelements. J. Mol. Evol. 50, 391–396. Kast, H.R., Nguyen, C.M., Sinal, C.J., Jones, S.A., Laffitte, B.A., Reue, K., Gonzalez, F.J., Willson, T.M., Edwards, P.A., 2001. Farnesoid X-activated receptor induces apolipoprotein C-II transcription: a molecular mechanism linking plasma triglyceride levels to bile acids. Mol. Endocrinol. 15, 1720–1728. Kushwaha, R.S., Hasan, S.Q., McGill Jr., H.C., Getz, G.S., Dunham, R.G., Kanda, P., 1993. Characterization of cholesteryl ester transfer protein inhibitor from plasma of baboons (Papio sp.). J. Lipid Res. 34, 1285–1297. Luo, C.C., Li, W.H., Chan, L., 1989. Structure and expression of dog apolipoprotein A-I, E, and C-I mRNAs: implications for the evolution and functional constraints of apolipoprotein structure. J. Lipid Res. 30, 1735–1746. Mak, P.A., Laffitte, B.A., Desrumaux, C., Joseph, S.B., Curtiss, L.K., Mangelsdorf, D.J., Tontonoz, P., Edwards, P.A., 2002. Regulated expression of the apolipoprotein E/C-I/ C-IV/C-II gene cluster in murine and human macrophages. A critical role for nuclear liver X receptors alpha and beta. J. Biol. Chem. 277, 31900–31908. McGill Jr., H.C., McMahan, C.A., Kushwiha, K.S., Mott, G.E., Carey, K.D., 1986. Dietary effects on serum lipoproteins of dyslipoproteinemic baboons with high HDLI. Arteriosclerosis 6, 651–663. Pei, B., Sisu, C., Frankish, A., Howald, C., Habegger, L., Mu, X.J., Harte, R., Balasubramanian, S., Tanzer, A., Diekhans, M., Reymond, A., Hubbard, T., Harrow, J., Gerstein, M.B., 2012. The GENCODE pseudogene resource. Genome Biol. 13, R51. Puppione, D.L., Whitelegge, J.P., 2013. Proteogenomic review of the changes in primate apoC-I during evolution. Front. Biol. 8, 533–548. Puppione, D.L., Ryan, C.M., Bassilian, S., Souda, P., Xiao, X., Ryder, O.A., Whitelegge, J.P., 2010. Detection of two distinct forms of apoC-I in great apes. Comp. Biochem. Physiol. D Genomics Proteomics 7, 9–13. Raisonnier, A., 1991. Duplication of the apolipoprotein C-I gene occurred about forty million years ago. J. Mol. Evol. 32, 211–219. Scanu, A.M., Edelstein, C., 2008. HDL: bridging past and present with a look at the future. FASEB J. 22, 4044–4054. Shachter, N.S., 2001. Apolipoproteins C-I and C-III as important modulators of lipoprotein metabolism. Curr. Opin. Lipidol. 12, 297–304. Shachter, N.S., Zhu, Y., Walsh, A., Breslow, J.L., Smith, J.D., 1993. Localization of a liverspecific enhancer in the apolipoprotein E/C-I/C-II gene locus. J. Lipid Res. 34, 1699–1707. Shih, S.J., Allan, C., Grehan, S., Tse, E., Moran, C., Taylor, J.M., 2000. Duplicated downstream enhancers control expression of the human apolipoprotein E gene in macrophages and adipose tissue. J. Biol. Chem. 275, 31567–31572. Simonet, W.S., Bucay, N., Lauer, S.J., Taylor, J.M., 1993. A far-downstream hepatocytespecific control region directs expression of the linked human apolipoprotein E and C-I genes in transgenic mice. J. Biol. Chem. 268, 8221–8229.
