Nucleosome-like particles and acetylated histones occur near active promoters and enhancers, and certain transcription factors can recognize their target sites on the surface of a nucleosome in vitro; yet it has been unclear whether transcription factors can occupy target sites on nucleosomes in native chromatin. We developed a method for sequential chromatin immunoprecipitation of distinct nuclear proteins that are simultaneously cross-linked to nucleosome-sized genomic DNA segments. We find that core histone H2A co-occupies, along with the FoxA (hepatocyte nuclear factor-3) transcription factor, DNA for the albumin transcriptional enhancer in native liver chromatin, where the enhancer is active. Because histone H2A on nuclear DNA is only known to exist in nucleosomes, we conclude that transcription factors can form a stable complex on nucleosomes at an active enhancer element in vivo.The crystal structure of the nucleosome core particle reveals how the four core histone proteins within the histone octamer alter DNA structure and accessibility (1). Specifically, the DNA is bent and in some places kinked on the nucleosome (2, 3); histone binding occludes part of the DNA surface; and the nearly two wraps of DNA around the histone octamer place the double helices in close proximity. Although much research has focused on the ways that these parameters are inhibitory to transcription factor binding, DNA bending and interactions with histones could promote certain factors to bind nucleosomes, and DNA wrapping can bring cooperative sites together. Furthermore, targeting of chromatin-modifying complexes and other gene regulatory proteins (4, 5) to inactive, closed chromatin domains would appear to require prior site-specific DNA binding by factors that are not inhibited by nucleosome structure. We recently found that the FoxA (HNF3) 1 transcription factor, one of the first factors to bind the albumin gene in development (6), can bind its sites on nucleosomal DNA in vitro, regardless of histone acetylation state (7). However, whether factors such as FoxA can bind to nucleosomes in vivo is not clear and is the subject of this paper.If initial DNA binding factors are not inhibited by nucleosomes, might such factor-bound nucleosomes persist as parts of active gene regulatory complexes? Consistent with this idea, a variety of transcriptional enhancers have been found to exist in a short array of nucleosome-like particles in the cell types in which the enhancers are active (8 -13), including the albumin gene enhancer (14). Also, there are now many examples of chromatin immunoprecipitation data indicating that acetylated core histone proteins are in the vicinity of regulatory sequences to which transcription factors are bound. Furthermore, genome-wide studies in yeast showed that depletion of core histone proteins causes a decrease in expression of a significant fraction of genes (15), indicating that histones could contribute positively to their expression.In all such instances in which nucleosomes are not inhibitory to fact...

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Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver.Genetic analysis of hepatoma-derived cell lines revealed, many years ago, that expression of differentiated functions is regulated in trans by mechanisms whose final effects are negative (extinction) or positive (activation) (78). More recent studies of these hepatoma lines further showed that maintenance of the hepatic phenotype is an active process operating at the level of transcription (21,42). By analysis of DNA sequences implicated in liver-specific transcription, the identification and cloning of members of four major families of liverenriched transcription factors (LETF) have been achieved. These families, each characterized by structurally related DNA binding domains, include the hepatocyte nuclear factor families HNF1, HNF3, and HNF4 and the CCAAT enhancer binding protein (C/EBP) family (13,82). Determination of the tissue distribution of these factors and analysis of their hierarchical relations led to the hypothesis that the combinatorial action of LETF together with the ubiquitous transcriptional machinery of the cell is necessary and maybe even sufficient for the maintenance of liver-specific gene transcription (30, 81). Indeed, the H4IIEC3 differentiated rat hepatoma cells and their derivatives that stably express an extensive set of adult hepatic functions express all the LETF identified to date, while in the rare dedifferentiated rat hepatoma cells that have lost expression of all these hepatic functions, HNF4 and HNF1 are systematically absent. In addition,...