Cell fate specification relies on the action of critical transcription factors that become available at distinct stages of embryonic development. One such factor is NeuroD1, which is essential for eliciting the neuronal development program and possesses the ability to reprogram other cell types into neurons. Given this capacity, it is important to understand its targets and the mechanism underlying neuronal specification. Here, we show that NeuroD1 directly binds regulatory elements of neuronal genes that are developmentally silenced by epigenetic mechanisms. This targeting is sufficient to initiate events that confer transcriptional competence, including reprogramming of transcription factor landscape, conversion of heterochromatin to euchromatin, and increased chromatin accessibility, indicating potential pioneer factor ability of NeuroD1. The transcriptional induction of neuronal fate genes is maintained via epigenetic memory despite a transient NeuroD1 induction during neurogenesis. NeuroD1 also induces genes involved in the epithelial‐to‐mesenchymal transition, thereby promoting neuronal migration. Our study not only reveals the NeuroD1‐dependent gene regulatory program driving neurogenesis but also increases our understanding of how cell fate specification during development involves a concerted action of transcription factors and epigenetic mechanisms. Synopsis Using a genome‐wide approach this study delineates the binding requirements and target sites for NeuroD1, illustrating how this factor drives the formation of a transcriptional and epigenetic program to ensure neuronal differentiation. Genome‐wide assessment of NeuroD1 binding reveals its targeting to regulatory elements of critical neuronal development genes. Following expression, NeuroD1 locates to and binds its target genomic locations in a highly sequence‐specific fashion. Prior to NeuroD1 binding, its target sites are developmentally silenced by epigenetic mechanisms and regulatory factors. NeuroD1 binding initiates events that confer transcriptional competence to neuronal fate genes including conversion of heterochromatin to euchromatin and reprogramming of the transcription factor landscape. The transient action of NeuroD1 during neuronal development is sufficient to induce a neuronal gene expression program that is maintained by epigenetic mechanisms.

Chromatin immunoprecipitation (ChIP) is widely used to identify chromosomal binding sites. Chromatin proteins are cross-linked to their target sequences in living cells. The purified chromatin is sheared and the relevant protein is enriched by immunoprecipitation with specific antibodies. The co-purifying genomic DNA is then determined by massive parallel sequencing (ChIP-seq). We applied ChIP-seq to map the chromosomal binding sites for two ISWI-containing nucleosome remodeling factors, ACF and RSF, in Drosophila embryos. Employing several polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF-1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters. Further validation included controls using chromatin of mutant embryos that do not express ACF1 or RSF-1. Surprisingly, the ChIP-seq profiles were unchanged, suggesting that they were not due to specific immunoprecipitation. Conservative analysis lists about 3000 chromosomal loci, mostly active promoters that are prone to non-specific enrichment in ChIP and appear as ‘Phantom Peaks’. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin. Mining the modENCODE ChIP-seq profiles identifies potential Phantom Peaks in many profiles of epigenetic regulators. These profiles and other ChIP-seq data featuring prominent Phantom Peaks must be validated with chromatin from cells in which the protein of interest has been depleted.

Dosage compensation in male Drosophila relies on the X chromosome–specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC). The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called “high-affinity sites” (HAS). Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features. In sexually dimorphic species, unequal distribution of sex chromosomes requires adjustment of gene expression levels between the sexes. Male flies enhance transcription from the single X chromosome to meet the levels in females (XX). The specific recognition of sex chromosomes is a crucial step in this dosage compensation process. Intuitively, one might assume that sex chromosomes harbor distinct DNA sequence motifs for recruitment of the modulating machinery; however, no clearly defined motifs capable of fulfilling this role have yet been found. One explanation for this shortcoming could be our failure to date to identify a sufficiently large set of sites that serve as specific docking stations. In the following study, we have systematically mapped the strongest recruitment sites of the Drosophila dosage compensation complex (DCC) and identified shared sequence elements. The closer a gene resides to one of these sites the more robust is regulation by the DCC, which documents the function of our inventory of high-affinity binding sites.