Foxp3 + CD4 + CD25 + regulatory T (T reg ) cells are essential for the prevention of autoimmunity 1,2 . T reg cells have an attenuated cytokine response to T-cell receptor stimulation, and can suppress the proliferation and effector function of neighbouring T cells 3,4 . The forkhead transcription factor Foxp3 (forkhead box P3) is selectively expressed in T reg cells, is required for T reg development and function, and is sufficient to induce a T reg phenotype in conventional CD4 + CD25 − T cells [5][6][7][8] . Mutations in Foxp3 cause severe, multi-organ autoimmunity in both human and mouse 9-11 . FOXP3 can cooperate in a DNA-binding complex with NFAT (nuclear factor of activated T cells) to regulate the transcription of several known target genes 12 . However, the global set of genes regulated directly by Foxp3 is not known and consequently, how this transcription factor controls the gene expression programme for T reg function is not understood. Here we identify Foxp3 target genes and report that many of these are key modulators of T-cell activation and function. Remarkably, the predominant, although not exclusive, effect of Foxp3 occupancy is to suppress

We demonstrate that the binding sites for highly conserved transcription factors vary extensively between human and mouse. We mapped the binding of four tissue-specific transcription factors (FOXA2, HNF1A, HNF4A, HNF6) to 4,000 orthologous gene pairs in hepatocytes purified from human and mouse livers. Despite the conserved function of these factors, from 41% to 89% of their binding events appear to be species-specific. When the same protein binds the promoters of orthologous genes, approximately two-thirds of the binding sites do not align.Elements of transcriptional regulation have central roles in evolution [1][2][3] . In many cases, conserved biological processes are controlled by evolutionarily conserved regulatory programs while evolving phenotypes are associated with cross-species variation in transcription regulation 4 . However, in the absence of suitable genome-wide data, it is unclear what fraction of all protein-DNA interactions are under either positive or negative selective pressure 1 . A preliminary effort to compare genome-wide binding sites for two stem cell-specific transcription factors in human and mouse has suggested that large differences exist between mouse and human 5, 6 yet because the data were obtained using different To compare systematically the binding of transcriptional regulators to promoter regions across species, we designed carefully matched ChIP-chip experiments 7 in human and mouse. We created custom DNA microarrays that array ten kilobases of sequence surrounding the known transcription start sites of over 4,000 orthologous pairs of mouse and human genes. These genes were selected because their orthology could be unambiguously assigned and oligonucleotides could be designed to represent the putative regulatory regions at high density ( Figure 1A, Supplementary Methods). Forty-seven hand-curated, tissuespecific genes were included in the array design as controls.Chromatin immunoprecipitations were performed independently in primary hepatocytes directly isolated from mouse and human liver using antibodies against four tissue-specific transcription factors (FOXA2, HNF1A, HNF4A, HNF6) involved in liver development and regulation ( Figure 1B, Table S1) 7 . Hepatocytes were chosen as a representative tissue for these experiments because (1) they are functionally and structurally conserved among mammals 8 ; (2) their gene expression programs are similar across species (Table S1); (3) their gene expression patterns are largely unperturbed by isolation procedures 9 ; and (4) the transcription factors responsible for hepatocyte development and function are highly conserved 8 . We amplified and fluorescently labeled the DNA from these binding experiments, hybridized it to the microarrays, and then scored binding events 10 .Several possible outcomes can be distinguished when comparing a binding event in one species with the data from the second species (Figure 1). First, one can determine if a particular transcription factor binds anywhere within the arrayed region of the human ...

We mapped the transcriptional regulatory circuitry for six master regulators in human hepatocytes using chromatin immunoprecipitation and high-resolution promoter microarrays. The results show that these regulators form a highly interconnected core circuitry, and reveal the local regulatory network motifs created by regulator-gene interactions. Autoregulation was a prominent theme among these regulators. We found that hepatocyte master regulators tend to bind promoter regions combinatorially and that the number of transcription factors bound to a promoter corresponds with observed gene expression. Our studies reveal portions of the core circuitry of human hepatocytes.