Chromatin at Pu.1 cis-regulatory elements unfolds at the onset of hemangioblast formation and before the Csf1r promoter. Assay examining relative DNaseI accessibility of the Csf1r promoter (A) and the Pu.1 promoter (B,C) in ES cells, ES-derived cells, 3T3 fibroblast cells, CD41⁺ cells sorted from day-4 blast cell cultures, and macrophages (Mac). (C) LM-PCR looking at Pu.1 with a primer set hybridizing at greater distance from the transcription start. Naked DNA (DNA) served as control and sequences were annotated by an LM-PCR amplifying genomic DNA modified by a G-specific Maxam-Gilbert G reaction (G). For each experiment, equal DNaseI digestion of samples was confirmed by amplification with primers specific for rDNA genes. Regions of localized increases/decreases in DNaseI accessibility at Csf1r and Pu.1 are indicated as gray or white bars, respectively. Signal intensities were measured across the indicated regions and were calculated relative to the rDNA control, with ES cells set as 1. The nature of transcription factor binding sites and their position are indicated at the left. Note that chromatin opening was established in the absence of gene expression. This is further illustrated by the absence of increased DNaseI accessibility in the coding region compared with macrophages (C).