Global dynamics of stage-specific transcription factor binding during thymocyte development

Tomonori Hosoya,James Douglas Engel,Stephen C J Parker,Greggory Myers,Ricardo D’Oliveira Albanus,Jacob Kitzman,John Hensley,Yasuhiro Kyono

doi:10.1038/s41598-018-23774-9

Abstract

In vertebrates, multiple transcription factors (TFs) bind to gene regulatory elements (promoters, enhancers, and silencers) to execute developmental expression changes. ChIP experiments are often used to identify where TFs bind to regulatory elements in the genome, but the requirement of TF-specific antibodies hampers analyses of tens of TFs at multiple loci. Here we tested whether TF binding predictions using ATAC-seq can be used to infer the identity of TFs that bind to functionally validated enhancers of the Cd4, Cd8, and Gata3 genes in thymocytes. We performed ATAC-seq at four distinct stages of development in mouse thymus, probing the chromatin accessibility landscape in double negative (DN), double positive (DP), CD4 single positive (SP4) and CD8 SP (SP8) thymocytes. Integration of chromatin accessibility with TF motifs genome-wide allowed us to infer stage-specific occupied TF binding sites within known and potentially novel regulatory elements. Our results provide genome-wide stage-specific T cell open chromatin profiles, and allow the identification of candidate TFs that drive thymocyte differentiation at each developmental stage.

Highlights

T cells develop in the thymus, where biologically distinct events driven by the interplay of multiple transcription factors (TFs) acting in coordination take place at each thymocyte stage
ATAC-seq signals at the double positive (DP) stage reflected profiles that were similar to DNase-seq peaks of total adult thymocytes[22] (Supplementary Fig. S2), as anticipated
DP ATAC-seq peak signals were highly correlated with DNase-seq peak signals of total thymocytes

Summary

Introduction

T cells develop in the thymus, where biologically distinct events driven by the interplay of multiple transcription factors (TFs) acting in coordination take place at each thymocyte stage. Footprinting analysis[12,13] of those open chromatin regions revealed the high-resolution landscape of predicted TF-bound motifs within those sequences. Our ATAC-seq data enabled the discovery of both stage-independent and stage-specific domains of open chromatin, and the TF footprinting data revealed 10–20 novel protein bound sequences within the previously validated enhancers of the Cd4, Cd8, Trb and Gata[3] genes. Enrichment analyses of TF binding in stage-specific open chromatin allowed the identification of TF motifs potentially driving each stage of thymocyte development. These data demonstrate that stage-specific changes in open chromatin are highly dynamic as thymocytes develop and provide deep insight into how the stage-specific binding of multiple TFs orchestrate transcriptional regulatory networks

Methods

Results

Conclusion