Bouvardia Pascualii spec. nova (sect. Gymnosiphon, Rubiaceae) en Oaxaca, México

A new species of the genus Bouvardia belonging to the subgen. Bouvardiastrum, sect. Gymnosiphon was collected by José Pascual in the southern part of the state Oaxaca in Mexico. At first glance it is similar to the polymorphic B. multiflora (Cav.) Schult. et Schult. f. widely distributed in Mexico to El Salvador, but differs from it basically in having leaves with 3–5 veins, white flower and corolla tube glabrous on both sides

The RAI1 region 1523-1627 has the ability to bind nucleosomes and histones <i>in</i><i>vitro</i>.

<p>(<b>A</b>) The region of RAI1 with homology to the novel nucleosome binding region in SPBP binds HeLa nucleosomes independent of the histone tails. Intact nucleosomes isolated from HeLa cells were incubated with GST, GST-SPBP (1551-1666), and GST-RAI1(1523-1627) (left panel), and partial trypsin digested nucleosomes were incubated with GST, GST-GST-SPBP(ePHD), GST-SPBP(1551-1666), GST-RAI1(ePHD), GST-RAI1(PHD), GST-RAI1(1523-1627) and negative control GST-MLL(PHD3) (right panel) as indicated. Bound proteins were subjected to Western blotting using anti–histone H3 antibody and anti-GST antibody. (<b>B</b>) The novel nucleosome-binding region 1551-1666 of SPBP and 1523-1627 of RAI1 interacts with histones H2 and H3 variants. Labeled GFP-SPBP (1551-1666) and GFP-RAI1(1523-1627) were incubated with GST, GST-H2A, H2B, H3.1, H3.3 and H2AZ expressed and purified from <i>E. coli</i>. Interacting proteins were subjected to SDS-PAGE and visualized using a PhosphorImager. (<b>C</b>) Deletion of the novel chromatin binding region of RAI1 (1523-1627), or the ePHD domain of RAI1, has no significant impact on RAI1 distribution in the nucleus of HeLa cells. HeLa cells were transiently co-transfected with plasmid expressing mCherry-RAI1 and EGFP-RAI1 (Δ1523-1627) (upper panels), or mCherry-RAI1 and EGFP-RAI1(ΔePHD)(lower panels).</p

The F-boxes of SPBP and RAI1 interact with their atypical PHD fingers.

<p>(<b>A</b>) Schematic illustration of domain structures of human SPBP (1960aa) and RAI1 (1906aa). TAD: trans-activation domain, NLS: nuclear localization signals, DBD: DNA-binding domain, ePHD/ADD: extended plant homeodomain, Q1/Q2: glutamine-rich stretches, grey boxes A to G: represents evolutionary conserved regions with strong homology between SPBP and RAI1. (<b>B</b>) A cartoon depicting a model of the ePHD domain of SPBP. In this model the F box interacts with the PHD domain of SPBP to form an ePHD/ADD domain containing two interacting zinc-finger modules. The interleaved zinc-ligation topology of the PHD domain is shown. Abbreviations: Zn, zinc; C, cysteine; H, histidine; N, N-terminal end; C, C-terminal end. (<b>C</b>) A schematic presentation of the ePHD/ADD domain of SPBP is shown. Asterisks mark Cys and His residues that may serve as zinc ligands. The F box is indicated in dark grey. The N- and C-terminal amino acid positions demarcating the SPBP ePHD/ADD domain are shown. Truncated versions of the ePHD/ADD domains of SPBP and RAI1, containing zinc-ligands 3-12 interact with their own and the others F box, as revealed by two-hybrid analysis. The F boxes of both proteins were fused to GAL4-AD and tested towards GAL4-DBD constructs of SPBP(ePHD), SPBP(ePHD3-12), RAI1(ePHD3-12), MLL1(ePHD), MLL2(ePHD) and MLL3(ePHD). Interactions are indicated by growth on QDO medium. Yeast cell suspensions were spotted onto plates and allowed to grow at 30°C for 3 days. (<b>D</b>) Deletion- and point-mutational analysis of the ePHD/ADD domain of SPBP using the two-hybrid interaction system. SPBP (ePHD/ADD) (black), SPBP (ePHD3-12) (grey) and the PHD finger, named SPBP (ePHD5-12) (white) were fused to GAL4-DBD and tested for interaction with the F box of SPBP fused to GAL4-AD. In addition, three constructs obtained by site-directed mutagenesis, generating mutations in the putative zinc-ligands 1 and 2 in the F box of SPBP(ePHD/ADD) were fused to GAL4-DBD, and tested for interaction to the SPBP F box fused to GAL4-AD. Cysteine(s) are mutated to alanine(s) as indicated in (C).</p

The ePHD/ADD domains of SPBP and RAI1 are conserved in evolution.

<p>(A) Alignment of the putative ePHD/ADD domain of SPBP and RAI1 in mammalia, reptiles, birds, amphibians, fishes and insect. Cysteine and histidine residues that may serve as zinc-ligands are indicated by numbers above the alignment. The long loop region between zinc ligands 2 and 3 in all species is excluded from the alignment, but indicated above. The regions encompassing the putative GATA-1 like finger and the PHD finger are indicated above, while the Loop1, Loop2 and Loop3 indicated below are loop structures generally found in PHD fingers. The conserved Tryptophan units used to select blasted sequences are indicated by arrows, while amino acids probably involved in Smith-Magenis syndrome are encircled. Two of the RAI1 sequences (A.carolinensis and L.chalumnae) are truncated in the C-terminal end probably due to sequencing errors. The threshold for shading in manually refined Clustal W sequence alignment was set to 50% using BLOSUM 62 scoring matrix. (B) Phylogenetic tree of the ePHD/ADD domains of SPBP and RAI1 proteins. The phylogenetic tree was constructed by Mega5 software using the maximum likelihood method. Numbers in the branch represents the bootstrap values. Background color coding is used to represent species which possess SPBP (blue), RAI1 (pink) and uncharacterized SPBP/RAI1 like proteins (green). The phylogenetic tree is based on selected protein sequences from representative vertebrate organisms (e.g. Homo sapiens represent Mammalia). (C) The evolutionary tree of the ePHD/ADD domains of SPBP and RAI1 proteins. The alignment of all sequences is shown in Supporting information, Figure S1 and the protein sequence accession numbers are provided in the Supporting information, Table S3. </p

The novel nucleosome binding region of SPBP is conserved.

<p>Alignment of the novel nucleosome binding region of SPBP, and the corresponding region of RAI1, in human, lizard, frog, fishes and bird species. In ray-finned fish a 42 amino acid insertion (position 1923-1965) is omitted from the alignment. Position of AT-hook motif and a NLS signal is indicated above. The protein sequence accession numbers are provided in the Supporting information, Table S4, respectively.</p

Nucleosome positioning sequences impair the synergistic effect of NRF2 and SPBP on the p62 promoter.

<p>Reporter constructs (60 ng) containing the indicated nucleosome position sequences inserted downstream of the transcription start site of p62 promoter were transfected into HEK293 cells together with the indicated amounts of expression plasmids for SPBP and/or NRF2. The luciferase activity of the promoter constructs cotransfected with empty expression plasmids was set to 1. The data represent the mean of three independent experiments with standard deviations, each performed in triplicate (***p<0.001, **p<0.01, *p<0.05).</p

SPBP and NRF2 cooperate to induce expression from the p62 promoter, and colocalize in nuclear speckles.

<p>(<b>A</b>) SPBP and NRF2 cooperate to enhance expression from the wild type p62 promoter. Transient transfections were carried out in HEK293 cells using 60 ng of the p62 promoter construct (−1781/+46), and 50 ng or 100 ng of an NRF2 expression vector, and 100 ng of a SPBP expression vector, as indicated. The data represent the mean of three independent experiments with standard deviations, each performed in triplicate (***p<0.001, *p<0.05). (<b>B</b>) SPBP recruits NRF2 to specific nuclear speckles. HeLa cells were transiently transfected with expression vectors for EGFP-NRF2 and mCherry-SPBP, and analysed 24 hours post transfection by live cell imaging using a confocal laser scanning fluorescence microscope. The Pearson's colocalisation scatter was generated using Volocity (Perkin Elmer).</p

Sulforaphane enhances SPBP expression.

<p>(<b>A</b>) Schematic representation of the domain structure of human SPBP. TAD: trans-activation domain, DBD: DNA binding domain, NLS: Nuclear Localisation Signal, ePHD/ADD: extended PHD/ADD domain, Q1/Q2: Glutamine rich stretches. Numbers below indicate amino acid positions. (<b>B</b>) NRF2, SPBP and p62 display similar induction upon sulforaphane treatment. HeLa cells were exposed to 20 µM sulforaphane and cell extracts harvested for the indicated time points. Equivalent aliquots from the extracts were subjected to SDS-PAGE and western blot using specific anti-SPBP antibody, anti-p62 antibody, anti-NRF2 antibody or anti-actin as indicated. Fold induction calculated and correlated to actin in three independent experiments with standard deviations are shown to the right (**p<0.01, *p<0.05). (<b>C</b>) Control experiment showing that DMSO alone does not induce any changes in NRF2, SPBP or p62 expression levels. Equivalent aliquots of HeLa cell extracts exposed to DMSO and harvested for the indicated time points were subjected to SDS-PAGE and western blot using specific anti-SPBP antibody, anti-p62 antibody, anti-NRF2 antibody or anti-actin antibody as indicated. Fold induction calculated and correlated to actin are shown to the right.</p

SPBP mediates transcriptional activation via ARE elements.

<p>(<b>A</b>) Schematic representation of the human −1781/+46 p62 promoter construct in front of the Luciferase gene. Conserved transcription factor binding sites relevant for this study are indicated. (<b>B</b>) Mutation of the ARE elements impairs SPBP mediated activation of the p62 promoter. Transient transfections were carried out in HEK293 cells using 60 ng of wild-type or mutated reporter vectors, and 100 ng of expression vectors for SPBP (upper panel) or CBP (lower panel). The data represent the mean of three independent experiments with standard deviations, each performed in triplicate (**p<0.01, *p<0.05, <i>n.s.</i> not significant). (<b>C</b>) Chromatin immunoprecipitations show that SPBP is associated with the p62 promoter. HeLa cell extracts (1.5×10⁷ cells per antibody) were immunoprecipitated with preimmune serum, polyclonal anti-SPBP antibody or polyclonal anti-NRF2 antibody. Input Control (1:40) was included. PCR was performed on chromatin precipitated with each antibody using primers aligning to positions −1324/−1173 in the p62 promoter (upper panel). Primers aligning to positions −3351/−3069 of the cathepsin D promoter were used as control. (<b>D</b>) SPBP mediated enhancement of the NQO1 promoter is dependent on the ARE element. Transient transfections were carried out in HEK293 cells using 60 ng of wild-type or mutated reporter vectors, and 100 ng of SPBP expression vectors. The data shows the mean of three independent experiments with standard deviations, each performed in triplicate (**p<0.01, <i>n.s.</i> not significant).</p

Knock down of SPBP impairs p62 expression and sulforaphane induced p62 body formation.

<p>(<b>A</b>) Endogenous SPBP and p62 are coexpressed in several cell lines. Extracts of the indicated human cell lines were analysed by western blotting using the indicated antibodies. (<b>B</b>) siRNA mediated knock down of SPBP reduces p62 expression level. HeLa cells transfected with two various SPBP siRNAs were analysed by western blotting using antibodies as indicated (left panel). The graph (right panel) shows the fold reduction calculated and correlated to actin in three independent experiments with standard deviations (*p<0.05, <i>n.s.</i> not significant). (<b>C</b>) Knock down of SPBP reduces the amount of p62 mRNA transcripts. The p62 mRNA levels were measured by quantitative RT-PCR. Hela cells were transfected with SPBP siRNAs or Control siRNA. RT-PCR reactions were run on p62, GADPH and β-actin mRNA. The average amount of p62 mRNA correlated to β-actin and GADPH mRNA based on two independent experiments are shown with standard deviations (*p<0.05, <i>n.s.</i> not significant). (<b>D</b>) p62 body formation upon sulforaphane treatment is reduced in SPBP siRNA knock down cells. HeLa cells were transfected with SPBP siRNA or Control siRNA and treated with 20 µM sulforaphane for 8 hours two days post transfection. Cells were fixed, stained with polyclonal antibodies against SPBP (green) and p62 (red) and analysed by confocal microscopy. The graph shows counting of p62 bodies in cells, each based on counting of more than 60 cells. Arrowheads indicate some of the p62 bodies in the cytoplasm.</p