Foxa1-Specific Targets Are Enriched for p53 Binding Sites.

<p>(A) A set of Foxa1-only targets was defined as those sequences that had one or fewer sequence tags Foxa2 ChIP-Seq data set (yellow circle). Motif analysis of these sequences found in a weaker <i>forkhead</i> consensus. In addition to the <i>forkhead</i> consensus, several other motifs appeared in this set of sequences. The first (ACATG and ATG repeats with a spacer in the middle) comprises parts of the positional weight matrix (PWM) for p53. The second closely resembles the PWM of Klf12, also known as repressor of AP-2alpha (Tfap2a). Orthogonal analysis of sites bound only bound by Foxa1 (yellow circle) and gene expression changes in livers of Foxa1-deficient mice (light purple circle) shows that thirty-seven are direct targets of Foxa1. (B) Verification of Foxa1-only targets by qPCR. Filled bars, ChIP of liver chromatin with an anti-Foxa1 antibody, open bars, ChIP with an anti-Foxa2 antibody. Three negative control regions (Nc1, Nc2, and Nc3) with a low amount of reads are included Binding is expressed as enrichment of the PCR amplicon relative to input DNA in liver chromatin. (C) Binding of p53 to <i>cis</i>-regulatory elements of its previously identified targets (positive controls), the alpha-fetoprotein (AFP) and TNF receptor superfamily <i>Fas</i> genes and twelve additional Foxa1-only targets, including cell-cycle associated <i>Aatf</i> and <i>Zwint</i> genes in both wildtype (black bars) and Foxa1 mutant livers (white bars) (left panel) by ChIP of liver chromatin followed by qPCR. None of Foxa2-only sites tested were bound by p53 in wiltype livers (right panel) (D) Quantitative RT- PCR analysis for mRNA of Zwint, Zwint mRNA levels are significantly downregulated in livers of Foxa1 mutant mice by 30%, Values are represented as means plus standard error. P values were determined by Student's <i>t</i> test. * p-value<0.05 (E) Foxa1 targets are enriched in genes involved in cell differentiation, morphogenesis, movement, and cellular cycle. (F) A regulatory feed-forward loop involving Foxa1. In the set of Foxa1-specific targets, Foxa1 binds to regulatory elements of the nuclear receptor Esr2 (estrogen receptor), its co-activator Smyd3, and target gene Pgr (progesterone receptor). (G) Foxa1 only sites are more distributed more broadly surrounding TSS (+/−100 Kb) than dual Foxa1/Foxa2 targets (compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002770#pgen-1002770-g004" target="_blank">Figure 4E</a>).</p

Foxa2-Specific Targets Contain a Medium-Strength <i>Forkhead</i> Consensus and Control Genes in Steroid and Lipid Metabolism.

<p>(A) The motifs found by <i>de novo</i> analysis for both Foxa2-only bound set (yellow circle) resemble a <i>forkhead</i> binding site. Intersection of Foxa2-bound regions and genes differentially expressed in livers of Foxa2-deficient mice (light purple circle) identified three-hundred thirty-three direct targets. (B) Validation of Foxa2-only targets by qPCR. Filled bars, ChIP of liver chromatin with an anti-Foxa1 antibody, open bars, ChIP with an anti-Foxa2 antibody. Binding is expressed as enrichment of the PCR amplicon relative to input DNA in liver chromatin. Negative controls (Nc1, Nc2, Nc3) are regions with a low amount of reads. (C) Foxa2 targets are enriched in genes involved in lipid and steroid metabolism, protein modification, and carbohydrate metabolism. (D) Histogram of the distribution of Foxa2-only sites relative to TSS. Foxa2-only sites, similar to the Foxa1/Foxa2 dual targets (compare <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002770#pgen-1002770-g004" target="_blank">Figure 4E</a>), are distributed normally near transcription start sites (TSS), with most sites within ten kilobases (Kb) from TSS.</p

Foxa1 and Foxa2 Regulate Different Sets of Target Genes in the Adult Liver.

<p>(A,B) Expression of Foxa factors in reciprocal mutant mice. Black bar, wild-type liver, white bar, Foxa1-deficient liver, and grey bar, Foxa2-deficient liver. As expected, Foxa1 mRNA is undetectable in the Foxa1 Foxa1-deficient liver, while expression of Foxa2 is not changed in absence of Foxa1 compared to control mice on both the mRNA and protein level. Similarly, expression of Foxa2 is near background levels in Foxa2-deficient livers, while expression of Foxa1 is comparable to that in wild-type littermates. (C) Venn diagrams of the number of genes that are differentially expressed (|FC|> = 1.5 and FDR = 15%) in adult livers (total, downregulated, and upregulated) of Foxa1 (pink circles) and Foxa2 mutant (yellow circles) mice. (D) Confirmation of seven microarray targets changed in expression in the Foxa1-deficient liver by quantitative real-time PCR (qRT-PCR) (top panel). When the mRNA levels of the same genes were determined in the Foxa2-deficient liver (bottom panel), these targets were either not Foxa2-dependent at all or regulated in the opposite direction. Values are represented as means plus standard error. P values were determined by Student's <i>t</i> test. * p-value<0.05. (E) mRNA levels of previously identified Foxa2 targets in Foxa1 mutant mice. Values are represented as means plus standard error.</p

Evolution of Vertebrate Foxa Paralogs and Functional Redundancy in the Fetal Liver.

<p>(A) Phylogenetic tree of the Foxa subfamily of transcriptional regulators. The putative Foxa4 gene was lost in vertebrates during evolution. (B) Sequence alignment of mouse Foxa1 and Foxa2 proteins by ClustalW2 algorithm. The winged-helix DNA binding domain is highlighted in blue. ‘*’, identical residues in all sequences, ‘:’ highly conserved amino acids; ‘.’ weakly conserved amino acids. (C) Venn diagram of the number of genes that are differentially expressed in fetal livers of Foxa1 and Foxa2 single mutants, as well as the double mutant (shaded in blue) on embryonic day 18.5. The number of genes dependent on each single factor is small compared to the number of genes that are differentially expressed in the double mutant.</p

Dual Foxa1/Foxa2 Sites Contain a Perfect <i>Forkhead</i> Motif.

<p>(A) All sequences bound by both Foxa1 and Foxa2 in the liver contain at least one perfect match to the Foxa consensus (a 7-mer of 4 possible sequences, with variation in the second and fifth nucleotide, (T[A/G]TT[G/T]AC)), and often an additional Foxa-like motif, containing one or two degenerate nucleotides. Surprisingly, these sites show additional preferences for specific bases at positions flanking the <i>forkhead</i> motif, with a “C” occurring frequently one nucleotide upstream, and a “T” enriched immediately downstream of the core consensus sequence. (B) Confirmation of several common Foxa1/Foxa2 targets by quantitative RT-PCR and three negative control regions (with low amount of reads, Nc1, Nc2, and Nc3). Binding is expressed as enrichment of immunoprecipitated DNA relative to input DNA in liver chromatin. (C) Functional categories over-represented in the genes bound by both Foxa1 and Foxa2 include ‘embryonic development’, ‘regulation of transcription’, and ‘lipid metabolism’. Biological pathways enriched for Foxa1/Foxa2 targets are ‘metabolism of xenobiotics’, ‘vesicular transport’ and ‘receptor tyrosine kinase signaling’. (D) Foxa1 and Foxa2 bind to four distinct intronic regions of <i>Fto</i> (fat mass and obesity associated) gene, which is associated with the risk of diabetes. (E) Histogram of <i>cis</i>-regulatory elements bound by both Foxa paralogs shows a normal distribution near transcription start sites (TSS), with most sites within ten kilobases (Kb) from TSS.</p

Identification of Genomic Targets of Foxa1 and Foxa2 in Adult Liver.

<p>(A) Venn diagram showing the results of genome-wide location analysis for Foxa1 and Foxa2 in the adult liver, identifying 5,562 binding sites for Foxa1 and 11,097 for Foxa2, of which 3,120 were called bound by both factors by the GLITR algorithm (certain common targets). Many of the apparently Foxa1- or Foxa2-specific regions contained overlapping reads from the opposite factor, indicating that the sites may be common, but the reads did not reach significant depth to be called by GLITR. We defined more stringent criteria for Foxa1-only and Foxa2-only binding sites. The sets of unique targets (yellow circles) contain peaks for the first factor that include at most one tag per million per KB for the other factor (1,816 Foxa1 sites with one or no Foxa2 tag and 5,682 Foxa2 sites with one or no Foxa1 tag). (B) Foxa1 and Foxa2 can occupy common binding sites (top panel) in the adult liver, or sites specific to either factor (Foxa1-only binding site, middle panel; Foxa2-only binding site, bottom panel). (C) Both common and unique binding sites for Foxa1 and Foxa2 can co-occur at a single genomic locus. (D) Comparison of Foxa1 and Foxa2 binding sites to a profile of H3K3me1 regions (purple circle) in the adult mouse liver.</p

Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor

The bromodomain containing proteins TRIM24 (tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis, and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of <i>N</i>,<i>N</i>-dimethyl­benzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor <b>8i</b> (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC <i>K</i>_d = 31 nM and ITC <i>K</i>_d = 14 nM, respectively). With its excellent cellular potency (EC₅₀ = 50 nM) and favorable pharmacokinetic properties (<i>F</i> = 29%), <b>8i</b> is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo