GSE159960
Stage-specific action of Runx1 and GATA3 controls silencing of PU.1 expression in mouse pro-T cells
PU.1 (encoded by Spi1), an ETS-family transcription factor with many hematopoietic roles, is highly expressed in the earliest intrathymic T cell progenitors but must be downregulated during T-lineage commitment.  Transcription factors Runx1 and GATA3 have been implicated in this Spi1 repression, but the basis of the timing was unknown.  We show that increasing Runx1 and/or GATA3 downregulates Spi1 expression in pro-T cells, while deletion of these factors after Spi1 downregulation reactivates its expression.  Leveraging the stage-specificities of repression and transcription factor binding revealed an unconventional but functional site in Spi1 intron 2. Acute Cas9-mediated deletion or disruption of the Runx and GATA motifs in this element reactivates silenced Spi1 expression in a pro-T cell line, substantially more than disruption of other candidate elements, and counteracts the repression of Spi1 in primary pro-T cells during commitment.  Thus, Runx1 and GATA3 work stage-specifically through an intronic silencing element in mouse Spi1 to control strength and maintenance of Spi1 repression during T-lineage commitment. Overall design: ChIP-seq; 5-10M cells were fixed with 1 mg/ml DSG (Thermo Scientific) in PBS for 30 min at RT followed by an additional 10 min with addition of formaldehyde up to 1%, or with 1% FA formaldehyde for 10 min.  Five ug of anti-GATA3 mAbs (a mixture of 2.5 ug of sc-268, Santa Cruz and, 2.5 ug of MAB26051, R&D systems) were pre-bound to Dynabeads anti-Mouse Ig (Invitrogen) and then added to the diluted chromatin complexes in parallel aliquots.  Precipitated chromatin fragments were cleaned up using Zymo ChIP DNA Clean & Concentrator.  ChIP-seq libraries were constructed using NEBNext ChIP-Seq Library Preparation Kit (E6240, NEB) and sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt.