Reks SE et al. (2014), Cooperative activation of Xenopus rhodopsin tra...

XB-ART-48512

BMC Mol Biol 2014 Feb 06;15:4. doi: 10.1186/1471-2199-15-4.

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Cooperative activation of Xenopus rhodopsin transcription by paired-like transcription factors.

Reks SE , McIlvain V , Zhuo X , Knox BE .

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BACKGROUND: In vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx5. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. Cooperative action by these transcription factors activates rod-specific genes such as rhodopsin. However, a quantitative mechanistic explanation of transcriptional rate determinants is lacking. RESULTS: We investigated the contributions of various paired-like transcription factors and their cognate cis-elements to rhodopsin gene activation using cultured cells to quantify activity. The Xenopus rhodopsin promoter (XOP) has a bipartite structure, with ~200 bp proximal to the start site (RPP) coordinating cooperative activation by Nrl/LNrl-Crx/Otx5 and the adjacent 5300 bp upstream sequence increasing the overall expression level. The synergistic activation by Nrl/LNrl-Crx/Otx5 also occurred when XOP was stably integrated into the genome. We determined that Crx/Otx5 synergistically activated transcription independently and additively through the two Pax-like cis-elements, BAT1 and Ret4, but not through Ret1. Other Pax-like family members, Rax1 and Rax2, do not synergistically activate XOP transcription with Nrl/LNrl and/or Crx/Otx5; rather they act as co-activators via the Ret1 cis-element. CONCLUSIONS: We have provided a quantitative model of cooperative transcriptional activation of the rhodopsin promoter through interaction of Crx/Otx5 with Nrl/LNrl at two paired-like cis-elements proximal to the NRE and TATA binding site. Further, we have shown that Rax genes act in cooperation with Crx/Otx5 with Nrl/LNrl as co-activators of rhodopsin transcription.

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Species referenced: Xenopus
Genes referenced: crx ddx39a maf nrl rax rax2 rho

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	Figure 1. Highly conserved vertebrate RPP. (A) A schematic representation of the rhodopsin gene, which consists of 5′ upstream sequence (5′ US), the proximal promoter (RPP, black box), the transcription initiation site (+1) and 5 exons (white boxes). (B) Sequence logo representation of an alignment of 33 vertebrate proximal promoters. Approximately 200 bp around the NRE were aligned using CLUSTALW and logos created using WEBLogo3.0. The species used included seven primates, five rodents, one erinaceid, two lagomorphs, three ungulates, one camelid, one loxodonta, two carnivores, one cetacean, one tenrec, one megabat, two marsupials, one monotreme, one reptile, two birds and two Xenopus. Rhodopsin promoters from fish did not fit this alignment.
	Figure 2. Effect of 5′ flanking sequences on Xenopus rhodopsin promoter activity in transfected 293 cells. (A) Comparison of luciferase activity (relative light units, RLU) from lysates of HEK293 or HEK293T cells transfected with a promoter-less pGL2 or XOP(−503/+41) containing luciferase plasmid in the absence (−TF) or presence (+TF) of pCS2-hCrx and pCS2-hNrl (+TF). The fold stimulation of XOP by hCrx-hNrl is indicated in parentheses. Data are mean ± S.E.M. (n = 2-6) Promoter basal activity (RLU) is shown in parentheses. (B) Comparison of luciferase activity in 293 T cell lysates transfected with plasmids containing different lengths of XOP 5′ US sequence: -5361, -503 and −145 all having the same 3′ sequence at +41. Cells were co-transfected with either human hCrx-hNrl or Xenopus LNrl-Otx5. Data are presented as mean RLU ± S.E.M. (n = 2-6). Promoter basal activity (RLU) is shown in parentheses. (C) Schematic of reporter constructs with additional Xenopus genomic DNA to control for the effect of plasmid size. All plasmids included the rhodopsin proximal promoter (RPP) upstream of the luciferase gene (LUC). XOP (−5361) contained 5.3 kb 5′ US sequence, -503UP and -503DWN contained a 5.8 kb fragment of the rhodopsin structural gene 5′ or 3′ to the RPP, respectively. The dotted line represents the inserted 5.8 kb rhodopsin gene. (D) Comparison of luciferase activity directed by different XOP constructs (−5361/+41, -503/+41, -145/+41, -503UP, -503DWN) in the absence (−TF) or presence (+TF) of Otx5 and L-Nrl. Activities are presented in RLU. The solid line represents the activity of samples transfected with empty pGL2 vector alone. The dotted line is a reference line for XOP(−503) to aid comparison. Data are presented as mean ± S.E.M. (n = 2-12). Promoter basal activity (RLU) is shown in parentheses.
	Figure 3. Comparison of transcriptional activity of Xenopus rhodopsin promoters stably integrated into the genome of 293 cells. Stable cell lines with integrated XOP (−5361/+41)-luciferase (#2 and #4) or XOP (−503/+41)-luciferase (#10 and #11) DNA were transfected in the absence or presence of Otx5, LNrl, Otx5-LNrl or hCrx-hNrl, and assayed for luciferase activity. Activities are presented in RLU normalized to total protein (μg) and represent mean ± S.E.M. (n = 4).
	Figure 4. Mutational analysis of Pax-like cis-elements in the RPP: Effect on Otx5-LNrl activation. (A) Diagram indicating the location of the Ret1, BAT1, NRE, Ret4 and TATA box (TA) elements and the mutations (TT to GG) constructed in these sequences. The transcription start site is marked by +1. (B) Cells were transiently transfected with a plasmid containing a wild type promoter (XOP(−503/+41)) or one of the mutants (m1-m10) in the absence (No TF) or presence of Otx5-LNrl. Activities in cell lysates are presented as mean ± S.E.M (n = 6-8). Promoter basal activity (RLU) is shown in parentheses.
	Figure 5. Xenopus Rax family transcription factors co-activate the rhodopsin promoter with Otx5-LNrl. Comparison of luciferase activity in lysates from cells transfected with XOP(−503/+41) alone or individually with (A) Otx5, Rax1a, Rax1b, Rax2a, Rax2b; (B) Otx5 in combination with Rax1a, Rax1b, Rax2a or Rax2b; (C) LNrl in combination with Rax1a, Rax1b, Rax2a or Rax2b; (D) LNrl/Otx5 in combination with Rax1a, Rax1b, Rax2a or Rax2b. All activities are presented as mean RLU ± S.E.M. (n = 6).
	Figure 6. Sequences upstream of the rhodopsin RPP enhance co-activation by Xenopus Rax family transcription factors. Comparison of luciferase activity in lysates from cells transfected with XOP(−5361/+41) alone or individually with (A) Otx5, Rax1b, Rax2b or in combination; (B) LNrl in combination with Otx5, Rax1b or Rax2b; (C) LNrl-Otx5 in combination with Rax1a, Rax1b, Rax2a or Rax2b. All activities are presented as mean RLU ± S.E.M (n = 6).
	Figure 7. Mutational analysis of Pax-like cis-elements in the RPP: Effect on Rax co-activation. Comparison of luciferase activity in lysates from cells transfected with XOP(−503/+41) or RPP mutants m1-m10alone, with Otx5-LNrl or with Otx5-LNrl-Rax2b. Activities are presented as mean RLU ± S.E.M. (n = 6-8). Promoter basal activity (RLU) is shown in parentheses.
	Figure 8. Effect of Rax concentration on co-activation of the RPP. (A) Schematic diagram of the plasmids containing RPP (XOP(−145/+41) or RPP missing Ret1 (XOP(−128/+41). (B, C) Cells were transiently transfected with either XOP(−145/+41) or XOP(−128/+41), LNrl-Otx5 and various concentrations of Rax1b (B) or Rax2b (C). Activities are presented as mean RLU ± S.E.M. (n = 6).
	Figure 9. Model of Xenopus rhodopsin transcriptional activation. Nrl binds to the rhodopsin promoter via a highly conserved NRE site (TGCTGAnnC) that is immediately upstream of the TATA binding site. Crx binds independently to two adjacent sites (G/CTTA). This combination synergistically activates transcription with each Crx contributing equally to the overall activity. The Ret1 site (CCAATTA) mediates Rax protein binding to stimulate transcription.

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