SUMMARY Recent studies have demonstrated that MyoD initiates a feed-forward regulation of skeletal muscle gene expression, predicting that MyoD binds directly to many genes expressed during differentiation. We have used chromatin immunoprecipitation and high throughput sequencing to identify genome-wide binding of MyoD in several skeletal muscle cell types. As anticipated, MyoD preferentially binds to a VCASCTG sequence that resembles the in vitro selected site for a MyoD:E-protein heterodimer, and MyoD binding increases during differentiation at many of the regulatory regions of genes expressed in skeletal muscle. Unanticipated findings were that MyoD was constitutively bound to thousands of additional sites in both myoblasts and myotubes, and that the genome-wide binding of MyoD was associated with regional histone acetylation. Therefore, in addition to regulating muscle gene expression, MyoD binds genome-wide and has the ability to broadly alter the epigenome in myoblasts and myotubes.

Little is known about the molecular development and heterogeneity of callosal projection neurons (CPN), cortical commissural neurons that connect homotopic regions of the two cerebral hemispheres via the corpus callosum and that are critical for bilateral integration of cortical information. Here we report on the identification of a series of genes that individually and in combination define CPN and novel CPN subpopulations during embryonic and postnatal development. We used in situ hybridization analysis, immunocytochemistry, and retrograde labeling to define the layer-specific and neuron-type-specific distribution of these newly identified CPN genes across different stages of maturation. We demonstrate that a subset of these genes (e.g., Hspb3 and Lpl) appear specific to all CPN (in layers II/III and V-VI), whereas others (e.g., Nectin-3, Plexin-D1, and Dkk3) discriminate between CPN of the deep layers and those of the upper layers. Furthermore, the data show that several genes finely subdivide CPN within individual layers and appear to label CPN subpopulations that have not been described previously using anatomical or morphological criteria. The genes identified here likely reflect the existence of distinct programs of gene expression governing the development, maturation, and function of the newly identified subpopulations of CPN. Together, these data define the first set of genes that identify and molecularly subcategorize distinct populations of callosal projection neurons, often located in distinct subdivisions of the canonical cortical laminae.

The binding of transcription factors to specific DNA target sequences is the fundamental basis of gene regulatory networks. Chromatin immunoprecipitation combined with DNA tiling arrays or high-throughput sequencing-ChIP-chip and ChIP-seq-has produced many recent studies that detail the binding sites of various transcription factors. Surprisingly, data from a variety of model organisms and tissues have demonstrated that transcription factors vary greatly in their number of genomic binding sites, and that binding events can significantly exceed the number of known or possible direct gene targets. Thus, our current understanding of transcription factor function must expand to encompass what role, if any, binding might play outside of direct transcriptional target regulation. Here, we discuss the biological significance of genome-wide binding of transcription factors and present models that can account for this phenomenon. Regulatory networks and the core model of gene regulationThe complex interactions between multiple transcription factors and gene targets across various tissues, cellular contexts, and time points are termed `transcriptional regulatory networks' (Box 1). It has been stated that a truly thorough understanding of such interactions should theoretically explain how an organism is `computed' from its DNA [1]. The core model of gene regulation posits that transcription factors recruit a polymerase complex to the transcriptional start site [2]. Transcription factors initiate this by binding at nearby or distant DNA sequences and directly interacting with components of the polymerase complex or with complexes that indirectly mediate the polymerase interaction. In eukaryotes, the latter may include chromatin remodelers or modifiers that facilitate access or increase protein-protein affinities via histone modifications [3,4]. The simplest view of the core model would suggest that factor binding directly correlates with transcriptional regulation. However, numerous examples of the separate regulation of factor binding and transcriptional activation suggest otherwise [5][6][7]. For example, recent studies indicate that the sequence of the DNA binding site can induce conformational changes in the bound transcription factor that permits transcriptional regulation by subsets of a transcription factor family that can bind to similar sites [8,9].

Rhabdomyosarcomas are characterized by expression of myogenic specification genes, such as MyoD and/or Myf5, and some muscle structural genes in a population of cells that continues to replicate. Because MyoD is sufficient to induce terminal differentiation in a variety of cell types, we have sought to determine the molecular mechanisms that prevent MyoD activity in human embryonal rhabdomyosarcoma cells. In this study, we show that a combination of inhibitory Musculin:E-protein complexes and a novel splice form of E2A compete with MyoD for the generation of active full-length E-protein:MyoD heterodimers. A forced heterodimer between MyoD and the full-length E12 robustly restores differentiation in rhabdomyosarcoma cells and broadly suppresses multiple inhibitory pathways. Our studies indicate that rhabdomyosarcomas represent an arrested progress through a normal transitional state that is regulated by the relative abundance of heterodimers between MyoD and the full-length E2A proteins. The demonstration that multiple inhibitory mechanisms can be suppressed and myogenic differentiation can be induced in the RD rhabdomyosarcomas by increasing the abundance of MyoD: E-protein heterodimers suggests a central integrating function that can be targeted to force differentiation in muscle cancer cells.[Keywords: E2A; Musculin; MyoD; myogenesis; rhabdomyosarcoma] Supplemental material is available at http://www.genesdev.org.