T cell lineage choice and differentiation in the absence of the RNase III enzyme Dicer

Abstract

The ribonuclease III enzyme Dicer is essential for the processing of micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) from double-stranded RNA precursors. miRNAs and siRNAs regulate chromatin structure, gene transcription, mRNA stability, and translation in a wide range of organisms. To provide a model system to explore the role of Dicer-generated RNAs in the differentiation of mammalian cells in vivo, we have generated a conditional Dicer allele. Deletion of Dicer at an early stage of T cell development compromised the survival of alphabeta lineage cells, whereas the numbers of gammadelta-expressing thymocytes were not affected. In developing thymocytes, Dicer was not required for the maintenance of transcriptional silencing at pericentromeric satellite sequences (constitutive heterochromatin), the maintenance of DNA methylation and X chromosome inactivation in female cells (facultative heterochromatin), and the stable shutdown of a developmentally regulated gene (developmentally regulated gene silencing). Most remarkably, given that one third of mammalian mRNAs are putative miRNA targets, Dicer seems to be dispensable for CD4/8 lineage commitment, a process in which epigenetic regulation of lineage choice has been well documented. Thus, although Dicer seems to be critical for the development of the early embryo, it may have limited impact on the implementation of some lineage-specific gene expression programs.

Figure 1.

Loss of Dicer activity. (a) Genomic PCR shows the Dicer ^lox allele in Dicer ^lox/lox B220⁺ lymph node B cells and DP thymocytes and in lckCre Dicer ^Δ/Δ B220⁺ lymph node B cells. Only the Dicer ^Δ allele is seen in lckCre Dicer ^Δ/Δ thymocytes from the DN4 stage onwards. White lines indicate that intervening lanes have been spliced out. (b) Loss of mature miR-181, miR-16, and miR-142s and accumulation of miR-181 and miR-16 pre-miRNAs in lckCre Dicer^Δ/Δ thymocytes. U6 small nuclear (snRNA) is a loading control.

Figure 2.

Reduced cellularity of lckCre Dicer ^Δ/Δ thymi, but no developmental block at the DN stage. (a) Thymocyte numbers and subset distribution defined by CD4 and CD8 expression in Dicer ^lox/lox and lckCre Dicer ^Δ/Δ littermates. The representation of thymocyte subsets and the total number of thymocytes are indicated. Note reduced cellularity in lckCre Dicer ^Δ/Δ thymi. (b) Expression of CD44 and CD25 on DN cells indicates normal DN subset distribution in lckCre Dicer ^Δ/Δ thymocytes. (c) Intracellular staining of DN thymocyte subsets indicates normal TCR-β expression in lckCre Dicer ^Δ/Δ DN thymocytes. (d) DNA content as assessed by propidium iodide (PI) staining indicates that lckCre Dicer ^Δ/Δ DN thymocytes proliferate normally. (e) TCR γδ-expressing cells are overrepresented in the absence of Dicer. (f) Genomic PCR shows that Dicer deletion is comparable, and virtually complete, in lckCre Dicer ^Δ/Δ γδ-expressing thymocytes.

Figure 3.

Increased cell death in the absence of Dicer. Thymocytes were stained for CD4, CD8, and DiOC6 as an indicator of mitochondrial membrane potential ex vivo or after culture. Histograms are gated on DP cells but not on light scatter.

Figure 4.

Transcriptional repression of centromeric satellite repeats and features of facultative heterochromatin are maintained in the absence of Dicer. (a) RT-PCR (controlled by GAPDH) did not detect transcripts of major and minor satellite repeat in wild-type (C57BL/10), Dicer ^lox/lox or lckCre Dicer ^Δ/Δ thymocytes, but did in differentiating myoblasts. White lines indicate that intervening lanes have been spliced out. (b) RNA FISH using Xist and Pgk probes of female (XX) control fibroblasts and Dicer ^lox/lox and lckCre Dicer ^Δ/Δ DP thymocytes (see Fig. S2) shows monoallelic, but not biallelic, expression of Pgk. Pgk signals did not overlap with Xist signals. (c) DNA from control XY cells and XX Dicer ^lox/lox or lckCre Dicer ^Δ/Δ thymocytes digested with Xba1/Nru1 (MeCp2) or Xba1/Mlu1 (Mtm1r), and probed for MeCp2 and Mtm1r CpG islands. Upper bands correspond to the methylated (inactive X) allele and lower bands to the unmethylated (active X) allele.

Figure 5.

CD4/CD8 lineage choice, lineage-appropriate gene expression, and developmentally regulated gene silencing in the absence of Dicer. Similar percentages of Dicer ^lox/lox and lckCre Dicer ^Δ/Δ thymocytes up-regulate CD5 and CD69 at the DP stage (a) and form transitional subsets that are defined by CD4 and CD8 expression (b, mean ± SD, n = 6, see Fig. 2 a), and the expression of CD69 and HSA follows the expected developmental sequence (c). (d) Real-time RT-PCR analysis of CD4 and CD8 lineage-specific transcripts in sorted DP, CD4 SP, and mature (TCR^hi) CD8 SP thymocytes normalized to UBC and YWHAZ control loci (mean ± SD, n = 3). (e) Real-time RT-PCR analysis of Tdt expression ex vivo, 10 h after phorbol ester and ionomycin stimulation (PMA+iono), or 10-h stimulation and 10-h recovery in fresh medium (normalized to UBC and YWHAZ, mean ± SEM, n = 2).

Figure 5.

CD4/CD8 lineage choice, lineage-appropriate gene expression, and developmentally regulated gene silencing in the absence of Dicer. Similar percentages of Dicer ^lox/lox and lckCre Dicer ^Δ/Δ thymocytes up-regulate CD5 and CD69 at the DP stage (a) and form transitional subsets that are defined by CD4 and CD8 expression (b, mean ± SD, n = 6, see Fig. 2 a), and the expression of CD69 and HSA follows the expected developmental sequence (c). (d) Real-time RT-PCR analysis of CD4 and CD8 lineage-specific transcripts in sorted DP, CD4 SP, and mature (TCR^hi) CD8 SP thymocytes normalized to UBC and YWHAZ control loci (mean ± SD, n = 3). (e) Real-time RT-PCR analysis of Tdt expression ex vivo, 10 h after phorbol ester and ionomycin stimulation (PMA+iono), or 10-h stimulation and 10-h recovery in fresh medium (normalized to UBC and YWHAZ, mean ± SEM, n = 2).