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. 2010 Jul 1;115(26):5376-84.
doi: 10.1182/blood-2010-01-263855. Epub 2010 Apr 21.

Gene enrichment profiles reveal T-cell development, differentiation, and lineage-specific transcription factors including ZBTB25 as a novel NF-AT repressor

Yair Benita  1 , Zhifang Cao, Cosmas Giallourakis, Chun Li, Agnès Gardet, Ramnik J Xavier
Affiliations

Gene enrichment profiles reveal T-cell development, differentiation, and lineage-specific transcription factors including ZBTB25 as a novel NF-AT repressor

Yair Benita et al. Blood. .

Abstract

The identification of transcriptional regulatory networks, which control tissue-specific development and function, is of central importance to the understanding of lymphocyte biology. To decipher transcriptional networks in T-cell development and differentiation we developed a browsable expression atlas and applied a novel quantitative method to define gene sets most specific to each of the represented cell subsets and tissues. Using this system, body atlas size datasets can be used to examine gene enrichment profiles from a cell/tissue perspective rather than gene perspective, thereby identifying highly enriched genes within a cell type, which are often key to cellular differentiation and function. A systems analysis of transcriptional regulators within T cells during different phases of development and differentiation resulted in the identification of known key regulators and uncharacterized coexpressed regulators. ZBTB25, a BTB-POZ family transcription factor, was identified as a highly T cell-enriched transcription factor. We provide evidence that ZBTB25 functions as a negative regulator of nuclear factor of activated T cells (NF-AT) activation, such that RNA interference mediated knockdown resulted in enhanced activation of target genes. Together, these findings suggest a novel mechanism for NF-AT mediated gene expression and the compendium of expression data provides a quantitative platform to drive exploration of gene expression across a wide range of cell/tissue types.

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Figures

Figure 1
Figure 1
Attributes of the enrichment score. (A) A heatmap representation of LIMMA linear coefficients for ES cells. The heatmap depicts linear coefficients derived from a pairwise comparison of expression values in ES cells and every other cell type/tissue in the panel. For illustration purposes only transcription factors are shown, are sorted vertically by the enrichment score (sum of coefficients in each row). (B) Expression values (black) and their standard deviation (black vertical lines), z-scores (red) and enrichment scores (yellow) for TBX21 and GAPDH. For illustration purposes only the top 10 and bottom 10 expressing tissues are shown. The left y-axis indicates expression values on log2 scale and z-score values. The right blue y-axis indicates enrichment scores. A distribution of enrichment scores was obtained from the highest score for each probe on the array and the percentiles of that distribution are shown in blue dotted lines.
Figure 2
Figure 2
Enrichment profiles of the top 10 tissues for 4 transcription factors. POU5F1, FOXP3, PHF7, and ATF5 are known for their role in embryonic stem cells (ES cells), T regulatory cells, testes, and liver, respectively. Bars are color-coded for the enrichment score relative to the distribution of highest and lowest score per gene. The values in the color scheme on top represent the percentiles of these distributions.
Figure 3
Figure 3
Transcription factors in T-cell development. (A) Enrichment profiles of 6 selected TFs across T-cell development stages (see supplemental Figure 2 for all TFs). These TFs score above the 97.5th percentile (indicated by a horizontal dashed line) in at least one development stage and their change across the development is larger than the 85th percentile. (B) All genes scoring above the 97.5th percentile that change across development above the 85th percentile were clustered to 40 clusters using K-means. Here 3 representing clusters are shown (see supplemental Figure 3 for all clusters). (C) Transcription factor binding site analysis results for each of the 3 clusters shown in panel B. Position weight matrices are ranked by z-score and only the top 10 results in each are shown.
Figure 4
Figure 4
T-cell–enriched transcription factors. (A) A heatmap representation of enrichment scores for genes identified as highly enriched in T cells. Genes are ranked from highest to lowest T-cell score and top scoring BCL11B is the only TF highly enriched in T cells. (B) A histogram representation of the sum of enrichment scores in T cells for all genes. The top 2.5% genes of that distribution were accepted as significantly enriched and are shown in panel A. (C) Enrichment profile of ZBTB25 showing the top 20 enriched cells/tissues. (D) Effect of ZBTB25 depletion on TCR-stimulated NF-AT signaling. ZBTB25 knockdown Jurkat E-6 stable cells were electroporated with 8 μg of NF-AT–luc reporter and 1 ng of renilla-luc reporter. After 18 hours of electroporation, cells from each sample were dispensed into 3 equal aliquots with 1 mL of complete IMDM media with or without anti-CD3 plus anti-CD28 antibodies (1 μg/mL of each) or PMA (50 ng/mL) plus anti-CD28 antibody (1 μg/mL). After another 7 hours of incubation, cells were harvested and examined for luciferase activity. The experiment was done in triplicates. (E) Effect of ZBTB25 depletion on the induction of TCR stimulated NF-AT target genes. Indicated ZBTB25 knockdown Jurkat E-6 stable cells were incubated with or without anti-CD3 plus anti-CD28 antibodies (1 μg/mL of each) for 24 hours. Then 3 sets of 1 × 106 cells from each experimental condition were harvested independently for real-time RT-PCR analysis of indicated gene expression. The experiment was done in biologic triplicates.
Figure 5
Figure 5
Genes enriched in hematologic cancers. (A) Top 15 enriched genes in B cell lymphoma. (B) CD70 enrichment profiles showing the top 5 cells/tissues in the primary cell dataset (top) and cancer cell dataset (bottom). Treg enrichment was abolished in the presence of cancer derived tissues due to significantly higher expression in cells derived from B-cell lymphoma. (C) Top 15 enriched genes in T-cell leukemia cells after reducing the highest enrichment score in primary T cells for each gene. For illustration purposes, in cases where multiple probes for the same genes exist, only the higher scoring probe is shown.
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