A monoallelic-to-biallelic T-cell transcriptional switch regulates GATA3 abundance

Abstract

Protein abundance must be precisely regulated throughout life, and nowhere is the stringency of this requirement more evident than during T-cell development: A twofold increase in the abundance of transcription factor GATA3 results in thymic lymphoma, while reduced GATA3 leads to diminished T-cell production. GATA3 haploinsufficiency also causes human HDR (hypoparathyroidism, deafness, and renal dysplasia) syndrome, often accompanied by immunodeficiency. Here we show that loss of one Gata3 allele leads to diminished expansion (and compromised development) of immature T cells as well as aberrant induction of myeloid transcription factor PU.1. This effect is at least in part mediated transcriptionally: We discovered that Gata3 is monoallelically expressed in a parent of origin-independent manner in hematopoietic stem cells and early T-cell progenitors. Curiously, half of the developing cells switch to biallelic Gata3 transcription abruptly at midthymopoiesis. We show that the monoallelic-to-biallelic transcriptional switch is stably maintained and therefore is not a stochastic phenomenon. This unique mechanism, if adopted by other regulatory genes, may provide new biological insights into the rather prevalent phenomenon of monoallelic expression of autosomal genes as well as into the variably penetrant pathophysiological spectrum of phenotypes observed in many human syndromes that are due to haploinsufficiency of the affected gene.

Keywords: GATA3; T-cell development; biallelic transcription; monoallelic transcription.

Figure 1.

Reduced activity of Gata3 alleles results in reduced expansion of immature T cells and elevated expression of myeloid transcription factor PU.1. (A,B) DN4 stage thymocytes were isolated from Gata3^z/+ (○) or Gata3^+/+ (•) mice and then cocultured on OP9-DL1 feeder cells. Cells were harvested and stained for CD4 and CD8 coreceptors followed by flow cytometric analysis at the indicated time points (after 1–4 d of coculture initiation) for cell number (A) and apoptosis (by Annexin V staining) (B). The horizontal bar in each genotype panel represents the average value for each analyzed genotype group. Representative data are shown from one experiment examining three mice of each genotype; similar results were collected from at least six mice of each genotype in two independent experiments. (C,E) Quantitative RT–PCR (qRT–PCR) in staged T cells of wild-type (Gata3^+/+; •) or heterozygous mutant (Gata3^z/+; ○) mice. GATA3 (C) and PU.1 (E) mRNA abundance was quantified using the total reverse-transcribed product from 100 live-cell equivalents of RNA normalized to HPRT mRNA. Each circle represents an individual mouse, and the black bars represent the averages. (*) P < 0.05; (NS) not significant. The data summarize duplicate measurements of three to eight mice of each genotype from at least two independent experiments. (D) Intracellular GATA3 (filled curve) protein abundance in staged T cells from wild-type (Gata3^+/+) (top panel) and heterozygous mutant (Gata3^z/+) (bottom panel) thymi was monitored by flow cytometry. The open curve represents background (IgG) staining in each sample. Representative histograms are shown as characterized in at least three mice of each genotype.

Figure 2.

Gata3 is transcribed from only one allele in HSCs and ETPs and then switches to biallelic transcription in half of DN3b and later stage T cells. Adult bone marrow cells and thymocytes were isolated using conventional cell surface markers (Hosoya et al. 2009; Ku et al. 2012), hybridized in situ to fluorescently labeled probes to detect either GATA3 (green spots) or CD45 (red spots), and analyzed by RNA-FISH (Materials and Methods). Bar, 3 μm. Representative images are shown from at least four mice of each developmental stage from five independent experiments and are summarized in Table 2. The images shown were taken as a single focal plane, but Z-stack images were recorded to confirm that no overlapping dots were misrepresented as a single dot on the single-focal-plane images.

Figure 3.

The commitment to Gata3 monoallelic or biallelic transcriptional status is mechanistically stable. (A) A hypothetical model. The GATA3-eGFP fusion (Gata3^g; red) allele-expressing adult Gata3^g/+ ETP cells are not viable because the reduced hypomorphic GATA3 monoallelic activity is not sufficient to allow ETPs to survive and they are eliminated, since GATA3 is vital for early T-cell generation, while wild-type (Gata3⁺; blue) monoallelic-expressing cells survive and continue to develop. Later during T cell development, after the DN3b stage, the second allele is activated in about half of the surviving cells and expresses both hypomorphic (Gata3^g) and wild-type (Gata3⁺) alleles. (B) Expression of a GATA3-eGFP fusion protein. eGFP expression from Gata3^+/+ (open curve) and Gata3^g/+ (filled curve) heterozygotes was monitored by flow cytometry in immature thymocytes to track transcription from the Gata3^g allele (Hosoya et al. 2009). Numbers above the bars indicate the percentage of eGFP-positive cells compared with wild-type controls from the same ancestral population. These histograms are representative of data recovered for at least five mice of each genotype from three independent experiments. (C) Hypothetical gradual (left) versus bimodal (right) switching models. (D) Gata3 monoallelic cells expressing the Gata3^g allele survive and develop in the absence of the Gata3⁺ allele. Gata3^g/g homozygous hypomorphic mutant thymocytes express eGFP in a cell-autonomous manner. Gata3^g/g or Gata3^+/+ HSCs were recovered from fetal livers and adoptively transferred into sublethally irradiated adult mice. Sixteen weeks after HSC transplantation, thymocytes were analyzed for eGFP expression. The diagram presents representative histograms in which seven to eight recipients were examined for each genotype in two independent experiments. (E) DN4 stage thymocytes were sorted into eGFP-negative (eGFP⁻; open curve) or eGFP⁺ (filled curve) populations from Gata3^g/+ mice and then cocultured on OP9-DL1 feeder cells. The eGFP expression profiles depicted immediately after sorting (left panels) or after 4 d of coculture and development into DP stage cells (right panels) are shown. The diagrams are representative of a total of eight mice examined in two independent experiments.