Identification of an Intronic Regulatory Element Necessary for Tissue-Specific Expression of Foxn1 in Thymic Epithelial Cells - PubMed (original) (raw)

. 2019 Aug 1;203(3):686-695.

doi: 10.4049/jimmunol.1801540. Epub 2019 Jun 26.

Jennifer E Cowan 1, Yueqiang Wang 1, Yu Tanaka 2, Yongge Zhao 1, Benjamin Voisin 3, Michael G Constantinides 4, Keisuke Nagao 3, Yasmine Belkaid 4, Parirokh Awasthi 5, Yousuke Takahama 2, Avinash Bhandoola 6

Affiliations

Identification of an Intronic Regulatory Element Necessary for Tissue-Specific Expression of Foxn1 in Thymic Epithelial Cells

Brian M Larsen et al. J Immunol. 2019.

Abstract

The thymus is critical for the establishment of the adaptive immune system and the development of a diverse T cell repertoire. T cell development depends upon cell-cell interactions with epithelial cells in the thymus. The thymus is composed of two different types of epithelial cells: cortical and medullary epithelial cells. Both of these express and critically depend on the transcription factor Foxn1 Foxn1 is also expressed in the hair follicle, and disruption of Foxn1 function in mice results in severe thymic developmental defects and the hairless (nude) phenotype. Despite its importance, little is known about the direct regulation of Foxn1 expression. In this study, we identify a _cis_-regulatory element (RE) critical for expression of Foxn1 in mouse thymic epithelial cells but dispensable for expression in hair follicles. Analysis of chromatin accessibility, histone modifications, and sequence conservation identified regions within the first intron of Foxn1 that possessed the characteristics of REs. Systematic knockout of candidate regions lead us to identify a 1.6 kb region that, when deleted, results in a near total disruption of thymus development. Interestingly, Foxn1 expression and function in the hair follicle were unaffected. RNA fluorescent in situ hybridization showed a near complete loss of Foxn1 mRNA expression in the embryonic thymic bud. Our studies have identified a genomic RE with thymic-specific control of Foxn1 gene expression.

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Figures

Figure 1.

Figure 1.

TEC specific chromatin accessibility identifies a functional regulatory element. (A) 50,000 cTEC (CD45-EpCAM+Ly51+UEA-1-) and mTEC (CD45-EpCAM+Ly51-UEA-1+) were sorted for ATAC-Seq at the indicated ages. Conservation (PhastCons) tracks of placental and vertebrate animals are shown from the UCSC Genome Browser. Knockouts of the first intron were done by CRISPR-Cas9. The resulting knockouts are indicated by the gray boxes and regions are identified as A and B. Mice resulting from the deletion of the A or B regions were designated Foxn1 ΔA and Foxn1 ΔB respectively. The gray bar represents the 28kb of DNA used to drive reporter expression in TECs in a previously described transgenic mouse (29). (B) Gross fur coat morphology of intron knockouts aged 6–8 weeks and Foxn1 nu/nu mice. (C) Images of the thymus of intron knockout mice and the Foxn1 nu/nu mouse. (D) 50,000 cells from the hair follicle (CD45-Sca-1-EpCAM+) were sorted for ATAC-Seq. The gray box indicates the deleted region in the Foxn1 ΔA/ΔA mouse. (E) Foxn1 expression in sorted hair follicle cells in anagen (4–5wk old mice) was measured by qRT-PCR. The bar plot represents mean ± SEM for n = 3–4 mice per genotype. ANOVA was performed to determine statistical significance.

Figure 2.

Figure 2.

Foxn1 ΔA/ΔA mice lack T cells. Flow cytometry was used to analyze T cell populations in the spleen as a readout of thymus function. (A) Representative flow cytometry plots of Lin- splenocytes comparing intron knockout mice to Foxn1 nu/nu mice. Numbers show the percentage of cells in each gate. Total numbers of CD19+ cells and TCRβ+ cells per spleen calculated from DAPI- live cells. (B) Representative flow cytometry plots showing DAPI-Lin- CD4+ and CD8+ populations in the spleen. Total number of CD4+ and CD8+ cells per spleen was calculated from DAPI- live cells. All bar plots show mean ± SEM for n = 4–7 mice (5–8weeks old) per genotype. ANOVA was performed to determine statistical significance. ** p < 0.01, *** p < 0.001.

Figure 3.

Figure 3.

Foxn1 ΔB/ΔB mice have mild mTEC defects but no apparent defect in T cell populations or cell numbers. Absolute numbers of total TEC (A) and cTEC and mTEC (B). (C) Representative flow plots of CD45-EpCAM+Ly51-UEA-1+ gated mTECs showing MHCIIhiCD80+ mTEChi vs MHCIIloCD80- mTEClo and calculations of overall cell numbers. (D) EpCAM+CD45- cTEC (Ly51+UEA-1-) and mTEC (Ly51-UEA-1-) were sorted and Foxn1 expression was measured by qRT-PCR. (E) Immunofluorescence analysis of thymic sections from indicated mice for β5t (green), Aire (magenta), and UEA-1 (blue). Scale bars indicate 100 μm. (F) Flow cytometry analysis of Aire expression in mTEChi (EpCAM+CD45-UEA-1+Ly51-MHCIIhiCD80+). (G) Lin-TCRβ-CD4-CD8- gated representative flow cytometry plots and overall cell numbers for ETPs (KithiCD25-), DN2 (KithiCD25+), DN3 (Kit-CD25+), and DN4 (Kit-CD25-). (H) Lin- gated thymocytes showing CD4 and CD8 profiles and overall cell numbers for each population. (I) Flow cytometry of thymocytes showing CD5 expression on DAPI- DP, TCRβhi SP4 and TCRβhi SP8 cells (J) Flow cytometry analysis and quantification of Tregs (CD4+CD8-TCRβ+CD25+FoxP3+) in the thymus. All bar plots show mean ± SEM for n = 4–7 mice (6–8 weeks old) per genotype. ANOVA was performed to determine statistical significance. *p < 0.05, **p < 0.01.

Figure 4.

Figure 4.

Foxn1 ΔD/ΔD mice have severely reduced T cells in the periphery. (A) gRNAs were designed to target potential regulatory elements within the A region of the first intron identified by ATAC-Seq. ATAC-Seq data shown is of E17.5 cTEC and mTEC. The gray boxes indicate regions that were deleted within the A region. The resulting mice were designated Foxn1 ΔC, Foxn1 ΔD, and Foxn1 ΔE. (B) Representative flow cytometry plots showing Lin- gated CD19+ B cells and TCRβ+ T cells from the spleen. Total cell numbers were calculated from all DAPI- live cells per spleen. (C) Flow cytometric analysis of DAPI-Lin- CD4+ and CD8+ cells in the spleen. Total cell numbers were calculated from DAPI- live cells. Absolute cell numbers in the liver (D) and lung (E) of MAIT cells (CD45+Thy1.2+TCR_γ_δ-TCRβ+mMR1 5-OP-RU tetramer+), NKT (CD45+Thy1.2+ TCR_γ_δ-TCRβ+mCD1dPBS57 tetramer+), and _γ_δ T cell populations (CD45+Thy1.2+ TCR_γ_δ+ROR _γ_t+) V_γ_4+ and V_γ_6+ (CD45+Thy1.2+ TCR_γ_δ+ROR _γ_t+ V_γ_4-). All absolute cell counts are shown as mean ± SEM for n = 3–8 mice (4–8 weeks old) per genotype. ANOVA was performed to determine statistical significance. *p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5.

Figure 5.

Foxn1 expression is absent in the Foxn1 ΔD/ΔD embryonic thymus. Foxn1 mRNA was visualized using fluorescent in situ hybridization. (A) Sagittal sections were taken of E11.5 embryos for each genotype. (B) Sagittal sections were taken of E10.5 control and Foxn1 ΔD/ΔD embryos. (C) Transverse sections were taken of E16.5 embryos. ao = aorta, tr = trachea, es = esophagus. White dotted outline indicates the thymus in Foxn1 ΔD/ΔD mice.

Figure 6.

Figure 6.

The _Foxn1_D RE is specific to Foxn1 and functions in cis. (A) Comparison of the mutations of the Foxn1 gene in Foxn1 ΔD/ΔD and Foxn1 nu/nu mice. (B) Images of Foxn1+/+, _Foxn1_ΔD/nu, and Foxn1 nu/nu mice showing normal overall fur coat in the _Foxn1_ΔD/nu mouse. (C) The _Foxn1_ΔD/nu mouse lacks a normal appearing thymus just like the Foxn1 nu/nu mouse. (D) Flow cytometric analysis was used to examine TCRβ+ cells in the spleen. Total cell numbers were calculated from DAPI- cells. There is a significant loss of TCRβ+ cells in the _Foxn1_ΔD/nu mouse however there is no difference in the overall number of TCRβ+ cells in the _Foxn1_ΔD/nu mouse compared to the _Foxn1_ΔD/ΔD and Foxn1 nu/nu mice. Total cell numbers were calculated from DAPI- live cells and shown as mean ± SEM for n = 3–4 mice (4–8 weeks old) per genotype. ANOVA was performed to determine statistical significance. *** p < 0.001.

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References

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