Regulatory elements and the complexity of identifying them


Gene Regulation
Img Src: Berger SL. 2000 Nature (doi:10.1038/35044160)

Understanding genetic variations that govern morphological differences is important in understanding the evolution of species (1). Although understanding mechanisms of gene regulation driving morphological differences is interesting, important, and necessary, it is incredibly difficult to investigate regulatory mechanisms because they are complex. First, body plans and body parts are highly complex and many genetic factors are responsible in development of body parts. Also, not all morphological differences are due to the same genetic mechanism. In this paper I will point out some of the problems in identifying genetic mechanisms governing morphological variations and discuss a few instances where regulatory elements have been identified.

Genetic factors regulating morphological differences
There are three potential mechanisms that can drive morphological differences. They are changes due to variation in coding regions of a gene resulting in a dysfunctional protein, variation in trans-regulatory proteins, and variations in cis-regulatory elements. It was clear by early 1970s that proteins may not explain most of the morphological differences. Based on various reports investigating polymorphisms in protein coding regions between humans and chimpanzee, Wilson and King concluded that regulatory elements and not proteins may be responsible for morphological variations (2).

Components of Regulatory System
Regulatory mechanisms have two components: trans-regulatory elements (TRE) and cis- regulatory elements (CRE). The scientific community is divided regarding which of the two contributes most to the morphological divergence. Some argue that although CRE have some role in evolution, biological processes such as speciation and adaptation are facilitated mostly by structural mutations (3, 4). It has been shown that a change in one amino acid can cause morphological changes such as coat color variation in mice (5). Many more examples of structural mutations contributing to morphological differences are discussed in ref.(4). There is also plenty of evidence that CRE can mediate morphological variations and many are summarized in ref.(6).

Cis- and trans-mediators both regulate morphological changes
Even though there are compelling evidence that both cis and trans regulatory changes can drive morphological differences, cis mediated changes seems to have gained momentum over trans mediated changes. This is because of at least two reasons. The first is due to the structural differencs between the two regulatory elements. CRE are non-coding, non-pleiotropic with spatial and temporal function, and modular (7, 8). Since they are non-coding, they can sustain more mutations than structural proteins. Unlike proteins (especially transcription factors that mediate trans-regulation) that have more than one function, CRE are non-pleiotropic thus any mutation sustained by CRE probably influences only one particular trait which unless deleterious does not affect the overall fitness of an organism. Thus CRE can accumulate mutations these mutations can produce changes that can be selected for. Finally, CRE are modular and one or a few mutations in various modules may not affect its overall function (9).

On the other hand, although proteins have spatial and temporal expression, they are pleiotropic and a change in a protein can make it dysfunctional. If the protein is involved in more than one pathway, then all the pathways are dysfunctional and this has a major impact in the fitness of the organism. Hence the argument is that proteins may not be the major contributors in driving morphological variations. However, this conclusion is influenced by current findings that show not many proteins are different between two species but the caveat here is how difference is defined. Most studies have investigated changes in nucleotide or amino acid sequences of coding regions but changes due to post transcriptional modifications such as splice variants are not fully investigated. It is possible that different splice variants function differently. Furthermore, the conclusion that only CRE and not trans-regulatory proteins mediate morphological variation is not entirely true as some studies have shown that changes in proteins can mediate morphological variations (5). Finally, it has been shown that proteins can be modular sustaining many changes in the amino acid level that has enabled them to retain their ancestral function at the same time gain novel functions (10).

Identifying regulatory elements
Even though it is likely that both CRE and TRE play a role in driving morphological differences, it is really hard to identify either of the regulatory elements. Finding TRE is harder because it can be anywhere in the genome whereas finding CRE is relatively easier because it is somewhere in that particular chromosome. Also, trans-regulatory networks can be huge consisting of thousands of proteins and hundreds of pathways and we are just beginning to understand the interconnection of such proteins in such networks. Therefore, it is not hard to imagine that finding a TRE that can be anywhere within the abyss of a regulatory network is next to impossible. While finding trans-regulatory elements is harder, finding CRE should be a simple straightforward task because by definition, CRE is defined as “a segment of DNA that regulates transcription; such segments typically lie immediately 5′ of the start site of transcription, but are often discontinuous, and individual segments can reside within introns, 5′ and 3′ UTRs, or tens of kilobases on either side of the gene they regulate” (6). However, finding CRE is also not that easy because the very properties of CRE (non-coding, non-pleiotropic, and modular) make them difficult to find. To elucidate the difficulties in identifying CRE, I will focus on recent reports on three morphological traits (pigmentation and hair structure in flies and pelvic structure in stickleback fish).

CRE that regulates abdominal pigmentation in Drosophila
Jeong et al. investigated what appears to be a simple morphological trait: the difference in male specific black pigmentation in abdomen of two Drosophila sister species that diverged recently; between 0.15-0.5 Mya (11). D. yakuba flies show the male specific pigmentation pattern but D. santomea flies lack the black pigmentation in their abdomen. These flies are geographically distributed in close by areas with D. yakuba found in lower altitudes where as D. santomea found in higher altitudes in an island in West Africa (12).

The authors seem to have chosen this particular morphological difference because of two reasons. First, they had extensive knowledge of the genetic mechanism regulating abdomen pigmentation in various Drosophila species including D. santomea because they had previously demonstrated that a pigment associated gene called yellow in the X chromosome, which is regulated by ABD-B Hox gene, plays a role in pigmentation in many Drosophila species (13, 14). Second, Carbone et al. had previously shown that four loci acting as QTL with large effect are responsible for the intra-species differences in abdomen pigmentation between D. yakuba and D. santomea (15). Of these four QTL two were in X chromosomes. From their previous work, which included D. santomea, they had observed that none of the mechanisms shared by other Drosophila species seem to play a role in lack of pigmentation in D. santomea indicating that a novel mechanism must be responsible for the colorless phenotype in D. santomea. It was also known that another gene tan is also involved in abdominal pigmentation. Both genes regulate the black melanin pigmentation in the abdomen and lack of tan causes severe under pigmentation. Naturally, tan was suspected to be responsible for loss of pigmentation in D. santomea.

Despite the intensive knowledge of pigmentation in flies, Jeong et al. had to perform multiple crosses, construct multiple transgenic constructs to measure gene expression, and make transgenic flies with these and additional constructs to show that of the four QTL found by Carbone et al. (15), the major QTL is the tan gene on X chromosome. This demonstrates that the same phenotype can be governed by multiple mechanisms and even if the mechanism that governs a morphological variation is well characterized in one species, this same mechanism may not be responsible for the same variation in another despite they are closely related. This shows the complexity of identifying CRE responsible for morphological variations.

To show that the lack of pigmentation in D. sanomea is not due to dysfunctional tan protein and not due to trans mediated elements the authors had to perform another series of crosses, conduct sequencing, and make additional transgenic flies. Fortunately, tan transcription unit in D. melanogaster was already known which enabled them to investigate for the CRE in the transcription unit of D. melanogaster (16). After finding the CRE in melanogster they had to repeat their experiments in D. santomea. They made transgenic D. santomea flies with the CRE of D. melanogaster and observed restoration of pigmentation in D. santomea concluding that CRE in tan is responsible for lack of pigmentation in D. santomea. In a sense they were fortunate possibly due to short divergent time between these two species that the D. santomea TRE was able to recognize the CRE from D. melanogaster, which enabled the restoration of pigmentation upon addition of functional CRE. Interspecies CRE may not necessarily restore function as some transcription factors may have also changed since two species have diverged (melanogaster and erecta in ref. 9).

Although Jeong et al. demonstrate that CRE in tan is responsible for pigmentation, they were not able to identify the differences within the CRE that may be responsible for pigment loss in D. santomea. After aligning the CRE sequences from many members of D. santomea and many other Drosophila species they found that there were more variations within species than between species. This demonstrates the complexity of pinpointing the changes within a CRE even after it is identified. Also, it is important to acknowledge the Drosophila genome is compact. Identifying the CREs in humans may be even more complicated due to tremendously large human genome full of repeats.

CRE that regulates trichome patches in Drosophila
The complexity of identifying mechanisms of another morphological trait, differences in trichome patches in various species of Drosophila, was demonstrated by Sucena and Stern (17, 18) and McGregor et al. (17, 18) in two papers published seven years apart. They compared abdominal hair patterns in five Drosophila species and picked two closely related Drosophila species one of which has hairy abdomen (D. simulans) and the other has bald abdomen (D. sechellia) for further experiments. Using crosses rather than molecular biology techniques used by Jeong et al to map the CRE, they showed that the regulation of hair development in flies maps to a region in X chromosome near the ovo/shaven-baby (ovo/svb) gene (17). Seven years later McGregor et al. was able to locate the enhancer regions that regulate differences between two Drosophlila species and they also come up with a robust possible model of evolutionary path for hairlessness in D. sechellia (18).

CRE that regulates pelvis modification in freshwater sticklebacks
All of the above mentioned studies demonstrate complexity of identifying genetic factors that regulate morphological variations in Drosophila. However, the complexity of variation is not unique to flies and this mechanism is at least equally complex in vertebrates as demonstrated by pelvic structure in stickleback fish. Marine stickleback fish have pelvic spines that protect them from predators. These spines are absent in freshwater fish. Shapiro et al. investigated the genetic mechanism that control pelvic reduction by performing crosses between the freshwater and marine fish followed by myriad of molecular biology techniques including QTL mapping, sequencing, RT-PCR, and in-situ hybridization (19). Their QTL mapping results indicated that there are multiple genes that are involved in pelvic modification in sticklebacks of which Pitx1 seemed the most important. Like Carbone et al (15), Shapiro et al also found that more than one genetic factor is responsible in controlling morphological trait. Furthermore, Pitx1 and other QTL discovered in this study were different from that previously reported by the same group indicating that more than one mechanism may be involved in spine formation in these fish (20).

Like Drosophila regulatory mechanism in stickleback is complex and despite patiently performing many complicated experiments, the best Shapiro et al could do was to identify the genetic region responsible for pelvis modification but not identify it. They did show that the no changes in the coding regions of the Pitx1existed and that the protein assembled properly and maintained proper spatial expression in freshwater fish that lacked the spine. It took them another six years to identify the exact location of the CRE mediating the pelvis structure which was found in enhancer region of Pitx1 gene reintroducing which restored the pelvic spine (21). Similar to Jeong et al., they found that there were multiple deletions within the enhancer region of Pitx1 in freshwater fish from various geographical areas demonstrating that the loss of pelvic spine has occurred independently and multiple times in the evolutionary past. Also, similar to D. santomea, the trans network must have remained preserved in the stickleback to allow for spine restoration upon introduction of the functional CRE. This demonstrates that even in the absence of CRE, trans-regulatory network preserve their function, whether this is due to preservation of ancestral function despite acquiring novel ones remains unclear.

It takes a lot of time, patience, and money among other things to perform such comprehensive studies to elucidate mechanisms of gene regulation governing morphological variations. The lesson that we can learn from these studies is not whether CRE or TRE drive evolutionary changes but how complex regulatory systems are. The studies mentioned above are commendable in that despite tremendous difficulties they have shed light on some of the most complex genetic mechanisms. With advent of novel molecular biology tools hopefully more studies of such kind will unravel more of the hidden genetic factors that control gene regulation. Possibly computational tools can be developed to investigate the cryptic regulatory elements in the genome. The long journey of understanding gene regulation lies ahead and investigating both cis and trans components of gene regulation will elucidate major mechanisms that drive evolutionary processes.

References
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2. King, M.C. and Wilson, A.C. (1975) Evolution at two levels in humans and chimpanzees. Science, 188, 107-16.
3. Coyne, J.A. and Hoekstra, H.E. (2007) Evolution of protein expression: new genes for a new diet. Curr Biol, 17, R1014-6.
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5. Hoekstra, H.E., Hirschmann, R.J., Bundey, R.A., Insel, P.A. and Crossland, J.P. (2006) A single amino acid mutation contributes to adaptive beach mouse color pattern. Science, 313, 101-4.
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7. Ludwig, M.Z., Bergman, C., Patel, N.H. and Kreitman, M. (2000) Evidence for stabilizing selection in a eukaryotic enhancer element. Nature, 403, 564-7.
8. Ludwig, M.Z., Patel, N.H. and Kreitman, M. (1998) Functional analysis of eve stripe 2 enhancer evolution in Drosophila: rules governing conservation and change. Development, 125, 949-58.
9. Ludwig, M.Z., Palsson, A., Alekseeva, E., Bergman, C.M., Nathan, J. and Kreitman, M. (2005) Functional evolution of a cis-regulatory module. PLoS Biol, 3, e93.
10. Lynch, V.J., Tanzer, A., Wang, Y., Leung, F.C., Gellersen, B., Emera, D. and Wagner, G.P. (2008) Adaptive changes in the transcription factor HoxA-11 are essential for the evolution of pregnancy in mammals. Proc Natl Acad Sci U S A, 105, 14928-33.
11. Jeong, S., Rebeiz, M., Andolfatto, P., Werner, T., True, J. and Carroll, S.B. (2008) The evolution of gene regulation underlies a morphological difference between two Drosophila sister species. Cell, 132, 783-93.
12. Lachaise, D., Harry, M., Solignac, M., Lemeunier, F., Benassi, V. and Cariou, M.L. (2000) Evolutionary novelties in islands: Drosophila santomea, a new melanogaster sister species from Sao Tome. Proc Biol Sci, 267, 1487-95.
13. Jeong, S., Rokas, A. and Carroll, S.B. (2006) Regulation of body pigmentation by the Abdominal-B Hox protein and its gain and loss in Drosophila evolution. Cell, 125, 1387-99.
14. Wittkopp, P.J., True, J.R. and Carroll, S.B. (2002) Reciprocal functions of the Drosophila yellow and ebony proteins in the development and evolution of pigment patterns. Development, 129, 1849-58.
15. Carbone, M.A., Llopart, A., deAngelis, M., Coyne, J.A. and Mackay, T.F. (2005) Quantitative trait loci affecting the difference in pigmentation between Drosophila yakuba and D. santomea. Genetics, 171, 211-25.
16. True, J.R., Yeh, S.D., Hovemann, B.T., Kemme, T., Meinertzhagen, I.A., Edwards, T.N., Liou, S.R., Han, Q. and Li, J. (2005) Drosophila tan encodes a novel hydrolase required in pigmentation and vision. PLoS Genet, 1, e63.
17. Sucena, E. and Stern, D.L. (2000) Divergence of larval morphology between Drosophila sechellia and its sibling species caused by cis-regulatory evolution of ovo/shaven-baby. Proc Natl Acad Sci U S A, 97, 4530-4.
18. McGregor, A.P., Orgogozo, V., Delon, I., Zanet, J., Srinivasan, D.G., Payre, F. and Stern, D.L. (2007) Morphological evolution through multiple cis-regulatory mutations at a single gene. Nature, 448, 587-90.
19. Shapiro, M.D., Marks, M.E., Peichel, C.L., Blackman, B.K., Nereng, K.S., Jonsson, B., Schluter, D. and Kingsley, D.M. (2004) Genetic and developmental basis of evolutionary pelvic reduction in threespine sticklebacks. Nature, 428, 717-23.
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1 Comment

Filed under My Life My Thoughts

One response to “Regulatory elements and the complexity of identifying them

  1. Limits of sequence and functional conservation
    Len A Pennacchio & Axel Visel
    Nature Genetics 42, 557–558 (2010) doi:10.1038/ng0710-557

    “…Although sequence conservation has proven useful as a predictor of functional regulatory elements in the genome, the observations by [Kunarso et al. Nat. Genet. 2010] are a reminder that it is not justified to assume in turn that all functional regulatory elements show evidence of sequence constraint. It is noteworthy that whereas OCT4 binding and NANOG binding diverged between human and mouse ES cells, binding of CTCF was highly conserved. Thus, it is expected that other DNA-binding proteins and chromatin marks will fall into a spectrum from strong to weak conservation between these two species. The notion that some regulatory networks have substantially changed in evolution is also supported by recent independent observations of lineage-specific network rewiring in vertebrate preimplantation embryos and adult liver tissue….”

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