Ash2l interacts with Tbx1 and is required during early embryogenesis

Abstract

TBX1 encodes a DNA binding transcription factor that is commonly deleted in human DiGeorge syndrome and plays an important role in heart development. Mechanisms of Tbx1 function, such as Tbx1 interacting regulatory proteins and transcriptional target specificity, are largely unknown. Ash2l is the mammalian homolog of Drosophila Ash2 (absent small homeotic 2) and is a core component of a multimeric histone methyltransferase complex that epigenetically regulates transcription via methylation of histone lysine residues. We undertook an unbiased yeast two-hybrid screen to look for functionally relevant Tbx1-interacting proteins and report a physical and functional interaction between Tbx1 and Ash2l. Tbx1 interacts with Ash2l in both yeast and mammalian cells and Ash2l acts as a transcriptional co-activator in luciferase reporter assays. Expression analysis shows that Tbx1 and Ash2l have overlapping mRNA and protein expression patterns during development. By generating an Ash2l knockout mouse utilizing gene-trap technology, we show that although Ash2l heterozygous mice are normal, Ash2l-null embryos die early during gestation. Thus, Ash2l is required for the earliest stages of embryogenesis. Furthermore, our finding of a physical interaction between Tbx1 and Ash2l suggest that at least some functions of Tbx1 may be mediated by direct interactions with a histone methyltransferase complex.

Figure 1

Tbx1 physically and functionally interacts with Ash2l in yeast and mammalian cells. (a) Tbx1 fused to the GAL4 DNA-BD interacts with Ash2l fused to the GAL4-AD allowing for yeast survival. (b) Full-length Tbx1-V5 co-immunoprecipitates with full-length myc-Ash2l and the myc-Ash2l mutants Δ100–150 and Δ310–413, but not Δ6–290. Protein was extracted from transiently transfected JEG3 whole-cell lysates. Immunoprecipitations were performed with anti-V5 or control IgG and complexes were analyzed by Western blot using HRP-conjugated anti-myc antibody. (c) Full-length Tbx1-V5 co-immunoprecipitates with full-length myc-Ash2l and the myc-Ash2l mutants 1–310, 1–405 and SPRY del but not 1–145 or 413–516. Methods were identical to those described in (b). (d) Full-length myc-Ash2l co-immunoprecipitates with full-length Tbx1-V5 and the Tbx1-V5 mutants F137Y, G299S and Trunc (Tbx1delC; Stoller and Epstein33). Methods were identical to those described in (b). (e) Schematic representation of full-length and mutant Ash2l constructs used to map the Ash2l interaction domain. The ability (+) or inability (−) of full-length Tbx1 to immunoprecipitate the Ash2l proteins is indicated. (f) Transcriptional co-activation of a luciferase reporter by TBX1 and Ash2l. Transient transfection of JEG3 cells with Ash2l augments TBX1-dependent transcriptional activation in a dose-dependent manner. Results are normalized for transfection efficiency and are expressed as mean + SD of nine experiments. The statistical significance of differences between groups was analyzed by the paired Student’s t-test. BD, binding domain; AD, activation domain; HRP, horse radish peroxidase; SD, standard deviation

Figure 2

Ash2l is broadly expressed in the mid-gestation mouse embryo. (a) Parasagittal section of E9.5 embryo showing Tbx1 expression by in situ hybridization. (b) Parasagittal section of E9.5 embryo showing Ash2l expression by in situ hybridization. (c) Parasagittal section of E13.5 embryo showing Ash2l protein expression by immunohistochemistry. Head mesenchyme (HM), pharyngeal arch core mesoderm (CM), pharyngeal endoderm (open arrowhead), ventricle (V), atria (A), somite (S), aortic sac (AoS), dorsal aorta (DAo), pharyngeal region of foregut diverticulum (asterisk), origin of first and second aortic arch arteries (closed arrowheads), vertebra (Vr), lung (Lu) and liver (Lv) are indicated for reference. E, embryonic age

Figure 3

Generation of Ash2l gene-trap knockout mice. (a) Targeting strategy of Ash2l gene-trap. The ROSAFARY retroviral cassette inserted between Ash2l exons 1a and 1b. The expected sizes of the HindIII (H), PstI (P) and EcoRI (E) digests recognized by the indicated probe (**) are shown. (b) Confirmation of predicted Ash2l exon 1a-βgeo* splicing. RT-PCR shows the exon 1a-βgeo* species in heterozygous, but not wild–type (WT) Ash2l ES cells. An exon 1b-βgeo* species is not present in either heterozygous or WT cells. (c) Ash2l isoform 1a is predominant in ES cells. RT-PCR shows that the Ash2l 1a isoform is more highly expressed in WT cells compared with the 1b isoform. In heterozygous Ash2l ES cells, there is equal expression of the two isoforms. (d) Schematic representation of alternative splicing. Ash2l exons 1a and 1b splice with exon 2. In Ash2l gene-trap mice, exon 1a splices with βgeo*. Exon 1b-βgeo* splicing is shown for illustration. Primers used for RT-PCR are indicated. (e) Genomic Southern blot of WT (+/+) and targeted Ash2l gene-trap ES cells (+/−). WT (10.8, 6.4 or 10.8 kb) and mutant (1.5, 4.5 or 3.9 kb) bands are shown, respectively, for the HindIII, PstI and EcoRI restriction enzyme digests. RT-PCR, reverse transcription-polymerase chain reaction; ES, embryonic stem