Subtle Variation: Despite vast phenotypic differences, vertebrates have many readily recognizable specific cell types, like liver hepatocytes. The gene expression that defines specific cells depends on evolutionarily conserved orthologous transcription factors. Schmidt et al. (p. 1036 , published online 8 April) studied the conservation and divergence in the genome-wide binding of two such transcription factors, CEBPA and HNF4A, in livers from human, dog, mouse, short-tailed opossum, and chicken. Although the sequence bound by orthologous transcription factors was similar, the vast majority of binding events were unique to each species.

Cross-regulatory cascades between hepatic transcription factors have been implicated in the determination of the hepatic phenotype. Analysis of recruitments to regulatory regions and the temporal and spatial expression pattern of the main hepatic regulators during liver development revealed a gradual increase in complexity of autoregulatory and cross-regulatory circuits. Within these circuits we identified a core group of six transcription factors, which regulate the expression of each other and the expression of other downstream hepatic regulators. Changes in the promoter occupancy patterns during development included new recruitments, release, and exchange of specific factors. We also identified promoter and developmental stage-specific dual regulatory functions of certain factors as an important feature of the network. Inactivation of HNF-4α in embryonic, but not in adult, liver resulted in the diminished expression of most hepatic factors, demonstrating that the stability of the network correlates with its complexity. The results illustrate the remarkable flexibility of a self-sustaining transcription factor network, built up by complex dominant and redundant regulatory motifs in developing hepatocytes.

To mechanistically characterize the microevolutionary processes active in altering transcription factor (TF) binding among closely related mammals, we compared the genome-wide binding of three tissue-specific TFs that control liver gene expression in six rodents. Despite an overall fast turnover of TF binding locations between species, we identified thousands of TF regions of highly constrained TF binding intensity. Although individual mutations in bound sequence motifs can influence TF binding, most binding differences occur in the absence of nearby sequence variations. Instead, combinatorial binding was found to be significant for genetic and evolutionary stability; cobound TFs tend to disappear in concert and were sensitive to genetic knockout of partner TFs. The large, qualitative differences in genomic regions bound between closely related mammals, when contrasted with the smaller, quantitative TF binding differences among Drosophila species, illustrate how genome structure and population genetics together shape regulatory evolution.

We analyzed the ordered recruitment of factors to the human α ₁ antitrypsin promoter around the initial activation of the gene during enterocyte differentiation. We found that a complete preinitiation complex, including phosphorylated RNA pol II, was assembled at the promoter long before transcriptional activation. The histone acetyltransferases CBP and P/CAF were recruited subsequently, but local histone hyperacetylation was delayed. After transient recruitment of the human Brahma homolog hBrm, remodeling of the neighboring nucleosome coincided with transcription initiation. The results suggest that, at this promoter, chromatin reconfiguration is a defining step of the initiation process, acting after the assembly of the Pol II machinery.