A signature motif mediating selective interactions of BCL11A with the NR2E/F subfamily of orphan nuclear receptors

Despite their physiological importance, selective interactions between nuclear receptors (NRs) and their cofactors are poorly understood. Here, we describe a novel signature motif (F/YSXXLXXL/Y) in the developmental regulator BCL11A that facilitates its selective interaction with members of the NR2E/F subfamily. Two copies of this motif (named here as RID1 and RID2) permit BCL11A to bind COUP-TFs (NR2F1;NR2F2;NR2F6) and Tailless/TLX (NR2E1), whereas RID1, but not RID2, binds PNR (NR2E3). We confirmed the existence of endogenous BCL11A/TLX complexes in mouse cortex tissue. No interactions of RID1 and RID2 with 20 other ligand-binding domains from different NR subtypes were observed. We show that RID1 and RID2 are required for BCL11A-mediated repression of endogenous γ-globin gene and the regulatory non-coding transcript Bgl3, and we identify COUP-TFII binding sites within the Bgl3 locus. In addition to their importance for BCL11A function, we show that F/YSXXLXXL/Y motifs are conserved in other NR cofactors. A single FSXXLXXL motif in the NR-binding SET domain protein NSD1 facilitates its interactions with the NR2E/F subfamily. However, the NSD1 motif incorporates features of both LXXLL and FSXXLXXL motifs, giving it a distinct NR-binding pattern in contrast to other cofactors. In summary, our results provide new insights into the selectivity of NR/cofactor complex formation

PRAME associates with the Elongin BC complex.

<p>(<b>A</b>) SDS-PAGE and silver staining showing affinity capture of proteins from HL60 whole cell extracts (WCE) by immobilised GST or GST-PRAME proteins. GST and GST-PRAME proteins are indicated. Putative PRAME-specific bands are indicated and bands of approximately 12 kDa and 17 kDa were excised for mass spectrometry analysis. (<b>B</b>) Co-immunoprecipitation of PRAME with Elongin complex components. Whole cell extracts of HEK293 cells transfected with PRAME-FLAG-6xHis (or empty vector control) applied to anti-FLAG sepharose beads as described in Materials and Methods. After extensive washing, co-purified PRAME and E3 ubiquitin ligase complex components were detected by western blotting using specific antibodies as indicated. (<b>C</b>) GST-pulldown experiment showing binding of ELB and ELC proteins in HL60 whole cell extracts to GST or GST-PRAME proteins. The top panel is a Coomassie-stained gel showing the input whole cell extract, and the purified GST and GST-PRAME proteins. The lower panels are western blots revealing PRAME, ELB and ELC proteins bound to GST proteins. (<b>D</b>) GST-pulldown experiments revealing interactions of ³⁵ [S]-labelled <i>in vitro</i> translated human ELC (hELC), <i>C.elegans</i> ELC (wELC), <i>C.elegans</i> ELC (L47D-L49D-Y88D-Y91D) (wELC mutant) and <i>C.elegans</i> ELB proteins with GST or GST-PRAME. (<b>E</b>) Yeast two hybrid assays of LexA-PRAME interactions with GAL4 AD-fused human ELC (hELC) or <i>C.elegans</i> proteins (wELB, wELC, wELC mutant). Western blots of the HA-tagged elongin fusion proteins are also shown. Reporter activity is expressed as β-galactosidase activity normalised to amount of protein in the extracts. (<b>F</b>) Immunofluorescence staining showing subcellular localisation of endogenous ELC, ELB and CUL2 proteins in HL60 cells. (<b>G</b>) Immunofluorescence staining showing colocalisation of endogenous ELC and PRAME proteins in HL60 cells following treatment with LPS/IFNγ for 4 hours.</p

Binding of PRAME to histone H3.

<p>(<b>A</b>) Co-immunoprecipitation of endogenous histones with PRAME-FLAG-6xHis isolated from extracts of transfected HEK293 cells. Input lanes (left panels) show the presence of endogenous or FLAG-tagged proteins in extracts from cells transfected with PRAME-FLAG or empty vector. After IP with anti-FLAG antibody, immunoblots were performed with the antibodies indicated. (<b>B</b>) GST-pulldown experiment showing association of histones with GST or GST-PRAME proteins. Whole cell extracts of HL60 cells were incubated with immobilised GST or GST-PRAME proteins, and bound proteins separated by SDS-PAGE. Immunoblots were performed with specific antibodies to detect association of histones H2A, H2B, H3 and H4 with GST proteins. (<b>C</b>) Direct association of histones with GST or GST-PRAME proteins. Core histone preparations were incubated with GST beads. Following extensive washing, bound histones were separated by SDS-PAGE and revealed by western blotting using specific antibodies as indicated.</p

Mass Spectrometry Identification of PRAME Binding Proteins.

<p>GST-PRAME binding proteins were affinity purified as described in Materials & Methods. Bands of approximately 17 kDa and 12 kDa (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058052#pone-0058052-g003" target="_blank">Fig. 3A</a>) were excised for mass spectrometry analysis. MS/MS fragmentation data were used to search the human NCBI database using the MASCOT search engine. Probability-based Mowse scores are shown.</p

Transcriptional and translational regulation of <i>PRAME</i> by PAMPs/IFNγ.

<p>(<b>A</b>) RT-qPCR measurements of <i>PRAME</i> gene expression relative to control <i>GAPDH</i> in HL60 cells in response to treatment with different PAMPs including lipopolysaccharide (LPS), peptidoglycan (PGN), muramyl dipeptide (MDP), zymosan (ZYM) and mannan (MAN) either alone (PBS) or in combination with IFNγ. Numbers on the x-axis indicate the time in hrs post-treatment. qPCR quantifications were performed in triplicate and the data shown represents the mean of two independent experiments, with error bars indicating standard deviations. The data is presented as fold induction relative to levels obtained at 0 hr (baseline). (<b>B</b>) RT-qPCR experiment performed as in (A) showing effect of pre-treatment with actinomycin D (10 μg/ml) or PBS on induction of <i>PRAME</i> transcript in HL60 cells by LPS/IFNγ (1 hour). (<b>C</b>) Association of <i>PRAME</i> transcripts with polysomes in HL60 cells following treatment with LPS/IFNγ for 0 and 4 hrs. Following cycloheximide treatment and sucrose density centrifugation of HL60 cell lysates, gradients were fractionated with continuous monitoring at 254 nm, to generate polysome profiles (top panel). RNA extracted from polysome fractions was analysed by Northern blotting and the <i>PRAME</i> transcripts visualised by phosphoimager (middle panels) and quantified by densitometry (bottom panels). <i>β-actin</i> was used as a control probe.</p

PRAME localises to the Golgi network following LPS/IFNγ treatment.

<p>(<b>A</b>) HEK293 cells (upper panels) were transiently transfected with PRAME-EGFP (green) and stained with α-PRAME antibody (red) to confirm the identity of the overexpressed EGFP fusion protein. U2OS cells (lower panels) were cotranfected with GFP (green) and PRAME-FLAG (red). Merged images indicate the extent of coincidence of the EGFP and α-PRAME signals, and nuclear DNA is indicated (blue). The right hand panels are western blots showing detection of GFP or PRAME-EGFP proteins in whole cell extracts of transfected U2OS cells. (<b>B</b>) Immunostaining of endogenous PRAME in HL60 cells using α-PRAME antibody following treatment with PBS, LPS/IFNγ or PGN/IFNγ for 4 hrs. (<b>C</b>) Immunostaining of endogenous PRAME in U937 cells with α-PRAME following treatment with LPS/IFNγ for 0, 1 and 4 hrs. (<b>D</b>) HL60 cells treated with LPS/IFNγ for 4 hrs and immunostained with α-Golgi 58K (green) and α-PRAME (red). Merged images show the extent of colocalisation of both proteins. For immunofluorescence (A–D), nuclear DNA was stained using Hoechst 33258 and images were captured using a LSM510 confocal laser scanning microscope. (<b>E</b>) Immunostaining of endogenous PRAME in HL60 cells using α-PRAME antibody following treatment with PBS or LPS/IFNγ for 4 hrs. (<b>F</b>) Quantification (n = 60) of the percentage of cells in (E) containing PRAME cytoplasmic foci in treated cells or controls. (<b>G</b>) Immunostaining of endogenous PRAME in MCF-7 cells using α-PRAME antibody.</p