ChIP-Seq and RNA-Seq Analyses Identify Components of the Wnt and Fgf Signaling Pathways as Prep1 Target Genes in Mouse Embryonic Stem Cells

Abstract

The Prep1 (Pknox1) homeodomain transcription factor is essential at multiple stages of embryo development. In the E11.5 embryo trunk, we previously estimated that Prep1 binds about 3,300 genomic sites at a highly specific decameric consensus sequence, mainly governing basal cellular functions. We now show that in embryonic stem (ES) cells Prep1 binding pattern only partly overlaps that of the embryo trunk, with about 2,000 novel sites. Moreover, in ES cells Prep1 still binds mostly to promoters, as in total embryo trunk but, among the peaks bound exclusively in ES cells, the percentage of enhancers was three-fold higher. RNA-seq identifies about 1800 genes down-regulated in Prep1 -/- ES cells which belong to gene ontology categories not enriched in the E11.5 Prep1i/i differentiated embryo, including in particular essential components of the Wnt and Fgf pathways. These data agree with aberrant Wnt and Fgf expression levels in the Prep1 -/- ES cells with a deficient embryoid bodies (EBs) formation and differentiation. Re-establishment of the Prep1 level rescues the phenotype.

The flexibility of a homeodomain transcription factor heterodimer and its allosteric regulation by DNA binding

“…C) was used as ligand. The decameric TGANTGACAG sequence is most frequently bound in vivo by the PREP1:PBX1 complex in mouse embryo and mouse embryonic stem cells . Fluorescence polarization shows that recombinant PREP1 FL :PBX1 FL and PBX1 1‐308 :PREP1 1‐344 dimers bind DNA in a saturable way and that their dissociation constants ( K D ) are 10 n m and 37 n m, respectively (Fig.…”

A tale of TALE, PREP1, PBX1, and MEIS1: Interconnections and competition in cancer

“…Analysis in multiple cell types shows that only a fraction of the binding sites of Prep1 and Meis1 is conserved in all cells, most being cell‐type dependent. The general rules set in the E11.5 embryo trunk (and Penkov et al, unpublished) have been reinforced in the ChIP‐seq and RNA‐seq analyses on other cell types. Using the same previously employed stringent molecular and statistical criteria, MEFs and ES cells each display 3–4,000 Prep1 binding sites covering about 2,000 genes, part of which are cell‐type specific.…”

“…An in‐depth analysis of the DNA binding landscape (ChIP‐Seq analysis) of Prep1, Pbx1, and Meis1/2 was carried out in the whole trunk of the E11.5 mouse embryo and in embryonic stem (ES) cells, mouse embryonic fibroblasts (MEFs), pre‐adipocytes and has uncovered a set of general rules that can be applied to most TALE proteins.…”

New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA

“…The DNA sequence that was employed to measure binding to the HD, was based on ChIP-seq data on whole embryo trunk and several murine and human cell lines30313233. These studies have provided consensus sequences for DNA binding by PREP1-PBX1: the very frequent decameric (TGATTGACAG) and the less frequent octameric (TGATTGAT) oligonucleotides conform to the above consensus sequences.…”

“…MEIS-A and MEIS-B domains are PREP1 motifs required for heterodomerization with PBX1. Panel B: Consensus sequences for DNA binding by PREP1-PBX1: the very frequent decameric (PMH) and the less frequent octameric (PH) oligonucleotides303132, are both highlighted in red in the sequence. Control probes correspond to two sequences that do not contain any PBX1 orPREP1 binding site.…”