Conservation of DNA and ligand binding properties of retinoid X receptor from the placozoan Trichoplax adhaerens to human

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Journal of Steroid Biochemistry and Molecular Biology 184 (2018): 3-10, doi:10.1016/j.jsbmb.2018.02.010.Nuclear receptors are a superfamily of transcription factors restricted to animals. These transcription factors regulate a wide variety of genes with diverse roles in cellular homeostasis, development, and physiology. The origin and specificity of ligand binding within lineages of nuclear receptors (e.g., subfamilies) continues to be a focus of investigation geared toward understanding how the functions of these proteins were shaped over evolutionary history. Among early-diverging animal lineages, the retinoid X receptor (RXR) is first detected in the placozoan, Trichoplax adhaerens. To gain insight into RXR evolution, we characterized ligand- and DNA-binding activity of the RXR from T. adhaerens (TaRXR). Like bilaterian RXRs, TaRXR specifically bound 9-cis-retinoic acid, which is consistent with a recently published result and supports a conclusion that the ancestral RXR bound ligand. DNA binding site specificity of TaRXR was determined through protein binding microarrays (PBMs) and compared with human RXR. The binding sites for these two RXR proteins were broadly conserved (~85% shared high-affinity sequences within a targeted array), suggesting evolutionary constraint for the regulation of downstream genes. We searched for predicted binding motifs of the T. adhaerens genome within 1000 bases of annotated genes to identify potential regulatory targets. We identified 648 unique protein coding regions with predicted TaRXR binding sites that had diverse predicted functions, with enriched processes related to intracellular signal transduction and protein transport. Together, our data support hypotheses that the original RXR protein in animals bound a ligand with structural similarity to 9-cis-retinoic acid; the DNA motif recognized by RXR has changed little in more than 1 billion years of evolution; and the suite of processes regulated by this transcription factor diversified early in animal evolution.Support for AMT was provided by the Tropical Research Initiative and an Internal Research and Development Award from the Woods Hole Oceanographic Institution. AMR was supported by NIH award R15GM114740. JM was supported by NSF award 1536530 to AMR. DM-P, BF and FMS were supported by NIH award R01DK094707 to FMS

Identification of a binding motif specific to HNF4 by comparative analysis of multiple nuclear receptors.

Nuclear receptors (NRs) regulate gene expression by binding specific DNA sequences consisting of AG[G/T]TCA or AGAACA half site motifs in a variety of configurations. However, those motifs/configurations alone do not adequately explain the diversity of NR function in vivo. Here, a systematic examination of DNA binding specificity by protein-binding microarrays (PBMs) of three closely related human NRs--HNF4α, retinoid X receptor alpha (RXRα) and COUPTF2--reveals an HNF4-specific binding motif (H4-SBM), xxxxCAAAGTCCA, as well as a previously unrecognized polarity in the classical DR1 motif (AGGTCAxAGGTCA) for HNF4α, RXRα and COUPTF2 homodimers. ChIP-seq data indicate that the H4-SBM is uniquely bound by HNF4α but not 10 other NRs in vivo, while NRs PXR, FXRα, Rev-Erbα appear to bind adjacent to H4-SBMs. HNF4-specific DNA recognition and transactivation are mediated by residues Asp69 and Arg76 in the DNA-binding domain; this combination of amino acids is unique to HNF4 among all human NRs. Expression profiling and ChIP data predict ≈ 100 new human HNF4α target genes with an H4-SBM site, including several Co-enzyme A-related genes and genes with links to disease. These results provide important new insights into NR DNA binding

Effect of HR3 RNAi depletion on mRNA abundance levels of genes involved in 20E response in the fat body of <i>Aedes</i> female mosquitoes.

<p>Female mosquitoes were injected with 1 µg of dsHR3 or dsMal. Transcript levels of Vg, E74B, EcR-A, EcR-B, USP-A, USP-B, betaFTZF1-A, and betaFTZF1-B were quantified by means of qPCR during a time-course experiment that covered from previtellogenesis (PV) to 36 h PBM. Each time point represents the average (± SEM) of three groups of three FBs. Each sample was normalized to its internal control ribosomal protein-7 mRNA. Three independent replicates of the experiment were assayed with three different cohorts of mosquitoes. *Indicates statistical significance <0.05.</p

HR3-knockdown <i>Aedes</i> mosquitoes showed a delay in impaired vitellogenin (Vg) mRNA and Vg protein levels at the end of vitellogenesis in the fat body.

<p>(A–C) Female mosquitoes were injected with 1 µg dsHR3 or dsMal RNAi, as described in Materials and Methods. HR3 (A and B) and Vg (C) mRNA expression was measured using qPCR at the indicated time points after blood feeding (PBM). Point 0h represents 72h post-eclosion in female mosquitoes prior to blood feeding; this was used as a reference control. Data are expressed as fold induction relative to S7. Data (means <u>+</u> standard errors of the means) from three independent experiments are shown. *Indicates statistical significance <0.05. (B) Magnification scale of the 36-h and 42-h PBM time points from the same experiment shown in A to demonstrate details of the HR3 depletion at the end of the vitellogenic cycle. (C) Monitoring the Vg transcript abundance in the same experiments as in A. A high level of the Vg transcript was observed in FBs of HR3-depleted female mosquitoes at 36 h PBM showing a delayed high expression of the <i>Vg</i> gene. (D) Western blot analysis of Vg protein during several time points of the first gonadotrophic cycle in the fat body of female mosquitoes treated with dsMal or dsHR3 RNAi. A total of 0.01 fat body equivalents was loaded in each lane for SDS/PAGE. A mixture of nine monoclonal antibodies against Vg small subunit was used for Vg protein detection (upper panels). Lower panels with Comassie-stained SDS/PAGE gels used for the western blot experiment are presented as loading controls. Three independent replicates of the experiment were made.</p

A schematic representation of the 20E regulated events during the first vitellogenic cycle in the fat body of the mosquito <i>A. aegypti</i>.

<p>After a blood meal activation, a high level of 20E acts via EcRA/USPB heterodimer activating early genes, BrZ2, E74B and E75A, which synergistically activate late genes such as <i>Vg </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045019#pone.0045019-Chen1" target="_blank">[15]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045019#pone.0045019-Cruz1" target="_blank">[18]</a>. TOR, activated by amino acids and insulin, is essential for activating late genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045019#pone.0045019-Hansen1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045019#pone.0045019-GuliaNuss1" target="_blank">[6]</a>. TOR is also involved in inhibition of the programmed autophagy during vitellogenesis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045019#pone.0045019-Bryant1" target="_blank">[19]</a>. HR3 is inhibited by E75A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045019#pone.0045019-Cruz1" target="_blank">[18]</a>, but activated by EcRA/USPB ensuring its timely expression. At the termination time, lowering of the 20E titer results in repressive action of BriZ1 (not shown) and BrZ4 on late genes such as Vg <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045019#pone.0045019-Chen1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045019#pone.0045019-Zhu1" target="_blank">[17]</a>. In this study, we have shown that HR3 is involved in inhibition of late target genes expression (<i>Vg</i>). On the other hand, HR3 activates the EcRB/USPA heterodimer. EcR has been shown to repress TOR and activate autophagy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045019#pone.0045019-Bryant1" target="_blank">[19]</a>. We postulate that it is the EcRB/USPA heterodimer that is responsible for these functions mediating action of HR3; however, this link requires additional confirmation. HR3 acts as an activator of betaFTZ-F1 that in turn is essential for maintaining cyclicity of egg development. The green ovals – EcR/USP 20E heterodimeric receptor; blue boxes – genes that encode 20E regulated transcription factors (early genes); purple boxes – Target-of Rapamycin; black boxes – autophagy; orange boxes – late target genes. Green arrows depict activating effects and red lines - repressive effects. The basics of the scheme were adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045019#pone.0045019-Thummel1" target="_blank">[46]</a>for comparison reasons.</p

HR3 RNAi depletion impaired the programmed fat body autophagy at the end of the vitellogenic cycle.

<p>(A) Lysotracker and DAPI staining of adult female FBs, 36 h PBM, treated with 1 µg of dsMal or dsHR3 RNAi. Newly emerged female mosquitoes were injected, and five days later they received a blood meal. FBs were dissected 36 h PBM and incubated for 5 min in a solution containing 200 nM of LysoTracker Red DND-99 and 0.01 microg/microl of DAPI. The scale bar is 50 µm. (B) mRNA levels of two genes involved in fat body autophagy at the end of the first vitellogenic cycle in the fat body of Aedes female mosquitoes. qPCR was performed as described before using specific primers for <i>Atg8</i> and <i>Debcl</i> genes. Each sample was normalized to its internal control ribosomal protein-7 mRNA and three independent experiments were assayed. *Indicates statistical significance <0.05.</p

Effect of HR3 RNAi depletion on ovarian development in the second gonadotrophic cycle in <i>Aedes</i> female mosquitoes.

<p>A. Representatives of ovaries from iHR3, iMal and untreated wild type female mosquitoes at 24 h PBM. Note that in iHR3 female mosquitoes ovaries are mostly underdeveloped, or lack visible yolk mass (white). Ovaries from iMal mosquitoes were normally developed similar to those of the wild type untreated control mosquitoes. C. Number of eggs per female mosquito in the second gonadotrophic cycle. iMal - HR3 RNA depleted, iMal – dsRNA Mal-treated, and wt - wild type female mosquitoes. The experiment was repeated three times with different cohorts of mosquitoes. *Indicates statistical significance <0.05.</p

A delay in a timely shutdown of the TOR signaling in fat bodies of HR3-depleted <i>Aedes</i> female mosquitoes.

<p>(A) Western blot analysis of phospho-S6-Kinase (S6K-P) and S6-Kinase (S6K) proteins during the termination of vitellogenesis in the fat body of Aedes female mosquitoes. One fat body equivalent per lane was loaded in a SDS/PAGE gel. The experiment was done in triplicate. (B) Densitometry measurements of three independent experiments from A showing higher levels of S6K-P in dsHR3 mosquitoes. Relative units normalized to its corresponding S6K levels. *Indicates statistical significance <0.05. (C) mRNA levels of LK6-Kinase (LK6) during the first vitellogenic cycle in FBs of female mosquitoes treated with dsMal or dsHR3 RNAi. qPCR was done as previously described. *Indicates statistical significance <0.05.</p