Evolution of a novel subfamily of nuclear receptors with members that each contain two

DNA binding domains.

Wu W, Niles EG, Hirai H, LoVerde PT. BMC Evol Biol. 2007 Feb 23;7:27. PMID: 17319953

Presented by Sheena ScrogginsBioinformatics I- Fall 09

Terminology

• NR-Nuclear receptor-protein that causes cellular response when ligand binds.

• DBD-DNA binding domain-protein domain with motif that recognizes DNA, either a specific sequence or has general affinity to DNA.

• LBD-Ligand binding domain-domain that binds ligand

• Dimer-Complex of 2 protein monomers

Nuclear Receptors

• Regulate transcription by binding to the promoter region of gene by the DBD and activation or repression of mRNA synthesis through co-regulators bound to the LBD.

• Normally have single DBD with a LBD.

Structure of Nuclear Receptors

• A-B) N-terminal regulatory domain: highly variable in sequence between various nuclear receptors.

• C) DNA-binding domain (DBD): Highly conserved domain containing two zinc fingers

• D) Hinge region: flexible domain which connects the DBD with the LBD.

• E) Ligand binding domain (LBD): Moderately conserved in sequence and highly conserved in structure between the various nuclear receptors.. Contributes to the dimerization interface of the receptor and in addition, binds coactivator and corepressor proteins.

• F) C-terminal domain: Variable in sequence between various nuclear receptors.

http://commons.wikimedia.org/wiki/File:Nuclear_Receptor_Structure.png

Why these NR are different

• This study included 3 2DBD-NRs-Sm2DBDα, Sm2DBDβ and Sm2DBDγ. from Schistosoma mansoni.

• Functional domains of 2DBD-Nuclear receptors and sequence alignment of the LBD domain. A. Schematic representation of functional domains of 2DBD-NRs isolated from Schistosoma mansoni. hRORα (human RAR-related orphan receptor) is an example that shows the 'typical' modular structure of nuclear receptors, which contain an A/B domain, a C domain (DBD), a D domain (hinge) and an E domain (LBD). Three Sm2DBD NRs exhibit a novel modular structure with an AB domain, two tandem DNA binding domains (C1 and C2), a D domain and an E domain. Sm2DBDα possesses an F domain at the C-terminal end of the E domain.

Origin and Duplication

6.A. Deduction from traditional view of metazoan phylogeny. The common ancestor of 2DBD-NR originated from DBD duplication in a common bilaterian ancestor (red branch). The fact that 2DBD-NRs are present in both Acoelomates and Coelomates supports this view. Star indicates duplication event(s).

Origin and Duplication

• 6.B. Deduction from the molecular view of metazoan phylogeny. The common ancestor of 2DBD-NR might originate in a common Protostome ancestor (red branch). The fact that 2DBD-NRs are present in both Ecdysozoa and Lophotrochozoa supports this view. Another possibility is that 2DBD-NR might originate in a common ancestor of the Bilateria and was lost in the Deuteros-tome lineage after the split of Protostomes and Deuterostomes (blue branch)

Duplication of 2DBD-NRs

• Duplication of 2DBD-NRs. A. Maximum Likelihood phylogenetic tree derived from sequences in the first and the second DBD of 2DBD-NRs.. Support values for the tree were obtained by bootstrapping a 100 replicates and are indicated above each branch. Branches under the threshold value of 45 were shown as polytomies. Bayesian inference with the same methods as in Fig. 3 running 5 million generations. The PPs are shown below each branch or after the ML bootstrapping value separated by a slash.

Gene tree

• Duplication of 2DBD-NRs. Figure 7.B shows that the common ancestor gene of 2DBD-NRs underwent duplication after the split of the Arthropods, Molluscs and Platyhelminths. In Mollusks, the orthologue gene (γ) underwent a duplication giving rise to the α/β gene. In Platyhelminths, the orthologue gene (γ) underwent a duplication giving rise to a new gene, this new gene underwent a recent duplication to give birth to the two present genes (α and β genes). All three genes are present in the flatworm S. mansoni and the planarian S. mediterranea suggesting that the two rounds of 2DBD-NR duplication occurred in a common ancestor of the Platyhelminths.

Data Mining

• Data mining at NCBI found 2DBD-NRs in other species: 3 in flatworm Schmidtea mediterranea (Se),1 in flatworm Dugesia japonica (Dj), 2 in mollusk Lottia gigantea (Lg),and 1 in arthropod Daphnia pulex (Dp)

• The P-box sequence CEACKK is not found in any other NR, suggesting that a novel target DNA specificity may exist for the first DBD.

Data mining continued

The P-box sequence of the second DBD (CEGGKG) is identical to that of most members of NR I.

Dimers• NRs can regulate transcription as a homodimer or as a heterodimer with retinoid ×

receptor (RXR), another nuclear receptor.

http://www.ks.uiuc.edu/Research/pro_DNA/ster_horm_rec/dbd/

Protein Interactions

• To determine whether 2DBD-NRs may form dimers, the interaction of Sm2DBDα, SmRXR1 and SmRXR2 was evaluated in a yeast two hybrid system

• As a strong dimer interface is known to be located in the LBD, GST pull-down experiments were performed (Fig. 5B).

Yeast two hybrid assay

• A- Different ORFs are expressed as fusion proteins to either the GAL4 DNA-binding domain (GAL4-BD) or its activation domain(GAL4-AD). If the proteins encoded by the ORFs do not interact with each other, the fusion proteins are not brought into close proximity and there is no activation of transcription of the reporter gene containing the upstream GAL4-binding sites.

• B- If the ORFs encode proteins that interact with each other, the fusion proteins are assembled at the GAL4-binding site of the reporter gene, which leads to activation of transcription.

http://cmbi.bjmu.edu.cn/cmbidata/proteome/method/research05.htm

Yeast two hybrid assay

• Protein-protein interaction of Sm2DBDα. A. Yeast two hybrid assays show that S. mansoni 2DBD-NR (Sm2DBDα) can act as a homodimer but not as a heterodimer with SmRXR1 and SmRXR2. Yeast AH109 was transformed with co-transformats: 1) pGBK-Sm2DBDα-C-F/pACT-SmRXR1, 2) pGAD-Sm2DBDα/pAs-SmRXR1-C-F, 3) pGBK-Sm2DBDα-C-F/pGAD-Sm2DBDα, 4) negative control plasmid pSV40/plamin C, 5) positive control plasmid pSV40/p53, 6) pGBK-Sm2DBDα-C-F/pACT-SmRXR2, 7) pGAD-Sm2DBDα/pAS-SmRXR2. The results show that Sm2DBDα can form a homodimer but not a heterodimer with SmRXRs.

GST pull-down

GST pull-down assay tests interactions between a tagged protein or the bait (GST.) and another protein (prey). The bait protein, is immobilized on a glutathione affinity gel. The bait serves as the secondary affinity support for identifying new protein partners or for confirming a previously suspected protein partner to the bait. Protein-protein interactions can be visualized by SDS-PAGE.

http://www.piercenet.com/products/browse.cfm?fldID=CAD5D96F-FBD3-4247-90E7-C1065B9D5630

• B. GST pull down verified that the S. mansoni 2DBDα-E-F domain, in which the dimer inter-face is located, can form a homodimer in vitro

GST pull-down

Further Research

• To determine the importance of gene duplication in NR evolution, more invertebrate NR complements will need to be analyzed.

• F domain needs further study to understand function and importance.

• Further studies of 2DBD-NR gene subfamily may contribute to understanding of gene duplication as an evolutionary force and to the phylogeny of the Metazoa.

References

• Wu W, Niles EG, Hirai H, LoVerde PT (2004). "Evolution of a novel subfamily of nuclear receptors with members that each contain two DNA binding domains". BMC Evol Biol 7 (Feb 23): 27. doi:10.1186/1471-2148-7-27. PMID 17319953. - Article, Figures and descriptions

• http://cmbi.bjmu.edu.cn/cmbidata/proteome/method/research05.htm - Yeast 2 hybrid

• http://en.wikipedia.org/wiki/Nuclear_receptor - Nuclear receptors

• http://www.piercenet.com/products/browse.cfm?fldID=CAD5D96F-FBD3-4247-90E7-C1065B9D5630 -GST pull-down