A new Groucho TLE4 protein may regulate the repressive activity of Pax5 in human B lymphocytes

Abstract

During mouse B-cell development, Pax5 is an essential transcription factor that acts as an activator of B-cell-specific genes and as a repressor of alternative lineage fates. The repressive function is mediated by the recruitment of members of the Groucho co-repressor family. Using an RNA display approach, we have isolated a transcript, called QD, specifically expressed in human pro-B and pre-B cells, which is derived from the human Groucho TLE4 gene. The QD transcript contains the first four TLE4 exons and an intronic sequence 3' of exon 4, demonstrating that QD is a splice variant of TLE4. The putative resulting protein of 94 amino acids corresponds to approximately half of an N-terminal tetramerization domain. We also show specific expression of TLE4 transcripts in human B cells and of TLE4 proteins in B-cell nuclei. Moreover, we demonstrate that recombinant QD protein binds to the TLE4 Q domain and is able to abolish the TLE4/Pax5 interaction. Thus, QD could act as a negative regulator of TLE4 function, in early B-cell differentiation.

Figure 3

B-cell-restricted RNA expression of QD and TLE4 transcripts. (a) and (b) Semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) expression of QD (a) and TLE4 (b) transcripts after actin calibration. RT–PCR was performed using a 5′ oligonucleotide in the Q domain common to the TLE4 and QD transcript, and a 3′ oligonucleotide in the 3′-untranslated region of the QD cDNA or in the TLE4 GP domain (see Fig. 1b), leading to PCR products of 515 bp and 238 bp, respectively. The following cell lines were tested: pro-B, lanes 1 (BV173), 2 (JEA2) and 3 (Reh); pre-B, lanes 4 (LAZ) and 5 (Nalm6); B, lanes 6 (Daudi), 7 (Namalwa) and 8 (JY); monocytes, lanes 9 (HL60), 10 (THP1) and 11 (U937); T, lanes 12 (Hsb2) and 13 (Jurkat). Average values of the relative quantity of PCR transcripts for three separate experiments are shown. (c) A Northern blot from the cell lines indicated above was hybridized with the TLE4 (see Fig. 1b) and the actin probes, successively.

Figure 1

Characterization of the QD transcript. (a) Sequence of the QD transcript obtained from the Image clone 140044. The cDNA (250 bp) first isolated by the RNA display technique is in bold. The potential polyadenylation site and splicing AG donor site at the end of exon 4 (see Figure 2) are underlined. (b) Schematic representation of TLE4- and QD-spliced mRNA and proteins. The oligonucleotides (small boxes) and the probes used for testing RNA expression of TLE4 and QD transcripts are shown for each mRNA. The different domains of TLE4 and QD proteins are indicated together with the corresponding amino acid positions. Q, glutamine-rich domain; GP, glycine–proline-rich domain; CcN, central region with a nuclear localization motif; SP, serine–proline domain; WD40, WD repeat domain. The C-terminal amino acid sequence (78–94) of the QD protein that differs from the TLE4 protein is indicated in black.

Figure 2

TLE4 genomic organization and generation of the QD and TLE4 transcripts. Top: exon/intron organization of the TLE4 gene. Data are from the Sanger Centre chromosome 9 Mapping Group (accession numbers: AL 353813 and AL 445252). The size of the introns is indicated. Bottom: generation of QD and TLE4 transcripts from the TLE4 gene.

Figure 4

B-cell nuclei-restricted TLE4 protein expression. Representative distribution of the TLE4 protein in a nucleus of pro-B (JEA2), pre-B (Nalm6), B (Namalwa), myeloblast (HL60), and T (HsB2) cells, obtained by immunostaining with the anti-TLE4 antibody. ‘mAb control’ represents a B-lymphocyte nucleus from a preparation incubated without the primary anti-TLE4 antibody.

Figure 5

Analysis of QD, TLE4 and Pax5 protein interactions. (a) Schematic diagram of TLE4, QD and Pax5 fusion proteins. The different domains of each protein are indicated (PD, paired domain; OP, octapeptide; HD, partial homeodomain; TA, transactivation region; ID, inhibitory domain; also see Fig. 1 legend). (b) The QD recombinant protein interacts with the Q domain of TLE4. GST pull-down assays were used to analyse the interactions between in vitro-translated ³⁵S-labelled TLE4-ΔW (lanes 1 and 2) and ³⁵S-labelled QD proteins (lanes 3 and 4) and GST or GST-TLE4Q proteins bound to glutathione-sepharose. (c) The QD recombinant protein inhibits the TLE4/Pax5 interactions. Upper panel: GST pull-down assays were performed with ³⁵S-labelled hPax5 protein and GST (lane 1), GST-TLE4-ΔW (lanes 2–5) bound to glutathione-sepharose. The GST-TLE4-ΔW proteins were incubated with an increasing amount of His-QD proteins (0·5, 1 and 3 µg, lanes 3, 4 and 5, respectively). Lower panel: for three independent experiments the percentage of inhibition was calculated for each QD/TLE4 molar ratio.

Figure 6

Speculative model of TLE4 function inhibition by QD protein. The tetrameric co-repressive TLE4 active form postulated by Chen et al. could be disrupted by breaking one of the two leucine-zipper like (LZL) motifs present in the N-terminal Q domain. Potential dimeric co-repressive inactive forms could be generated by a Leu to Pro amino acid substitution at position 38 in the first LZL1, or by interaction with the QD proteins.