The recognition of specific DNA-binding sites by transcription factors is a critical yet poorly understood step in the control of gene expression. Members of the Hox family of transcription factors bind DNA by making nearly identical major groove contacts via the recognition helices of their homeodomains. In vivo specificity, however, often depends on extended and unstructured regions that link Hox homeodomains to a DNA-bound cofactor, Extradenticle (Exd). Using a combination of structure determination, computational analysis, and in vitro and in vivo assays, we show that Hox proteins recognize specific Hox-Exd binding sites via residues located in these extended regions that insert into the minor groove but only when presented with the correct DNA sequence. Our results suggest that these residues, which are conserved in a paralog-specific manner, confer specificity by recognizing a sequence-dependent DNA structure instead of directly reading a specific DNA sequence.

SUMMARY UHRF1 is an essential regulator of DNA methylation that is highly expressed in many cancers. Here, we use transgenic zebrafish, cultured cells and human tumors to demonstrate that UHRF1 is an oncogene. UHRF1 overexpression in zebrafish hepatocytes destabilizes and delocalizes DNMT1, causes DNA hypomethylation and Tp53-mediated senescence. Hepatocellular carcinoma (HCC) emerges when senescence is bypassed. tp53 mutation both alleviates senescence and accelerates tumor onset. Human HCCs recapitulate this paradigm, as UHRF1 overexpression defines a subclass of aggressive HCCs characterized by genomic instability, TP53 mutation and abrogation of the TP53-mediated senescence program. We propose that UHRF1 overexpression is a mechanism underlying DNA hypomethylation in cancer cells and that senescence is a primary means of restricting tumorigenesis due to epigenetic disruption.

Pisitkun T, Jacob V, Schleicher SM, Chou C-L, Yu M-J, Knepper MA. Akt and ERK1/2 pathways are components of the vasopressin signaling network in rat native IMCD. Am J Physiol Renal Physiol 295: F1030 -F1043, 2008. First published July 30, 2008 doi:10.1152/ajprenal.90339.2008.-Vasopressin regulates water excretion through effects on the renal collecting duct. Vasopressin signaling in the inner medullary collecting duct (IMCD) is mediated by V2 receptor occupation coupled to the generation of cyclic AMP. Here, we employ a "systems" approach to analysis of vasopressin signaling. The objective is to investigate roles of activation of the Akt and ERK1/2 MAP kinase pathways, as well as Ca 2ϩ mobilization, in IMCD cells isolated from rat kidney. The V2 receptor-selective vasopressin analog dDAVP increased the state of Akt activation (increased phosphorylation at T308 and S473) and decreased the state of ERK1/2 activation (decreased phosphorylation at T202 and Y204). Akt activation was blocked by an inhibitor of PI3K, LY294002. In microdissected IMCD segments, nonperiodic spike-like increases in intracellular Ca 2ϩ (FLUO-4) were accelerated by vasopressin. Chelation of Ca 2ϩ or calmodulin inhibition markedly decreased Akt phosphorylation. Decreased ERK1/2 phosphorylation was associated with a decrease in MEK1/2 phosphorylation and an increase in c-Raf phosphorylation at S259 (an inhibitory site). Based on the current findings integrated with previous findings in the IMCD, we now report a 33-node vasopressin signaling network involved in vasopressin regulation of IMCD function.aquaporin-2; phosphoinositide 3-kinase; cyclic AMP; calmodulin WATER TRANSPORT across the renal collecting duct is controlled by the peptide hormone vasopressin, largely through the long-and short-term regulation of the apical water channel aquaporin-2 (AQP2) (24). Long-term regulation refers to changes in total abundance of AQP2 over a period of days. Short-term regulation refers to changes in AQP2-mediated water transport over periods of minutes through regulation of trafficking of AQP2 vesicles to and from the apical plasma membrane. Beyond vasopressin's role in transport regulation, the growing recognition that vasopressin regulates proliferation of normal inner medullary collecting duct (IMCD) cells (2) as well as of renal cysts derived from collecting duct cells (36) or cysts formed from renal epithelial cells grown in culture (21) brings renewed interest to vasopressin signaling mechanisms in collecting duct cells.A preliminary vasopressin signaling network in the renal IMCD, based on proteomic analysis and the existing literature, has been published in our previous paper (29) (see DISCUSSION for full details). The effects of vasopressin are a consequence of binding of vasopressin to basolateral V2 receptors. Although other processes have been proposed (22), many of the vasopressin effects are believed to be consequent to activation of the heterotrimeric G protein G␣s (GNAS) and to increases in intracellular Ca 2ϩ via calmodulin (8). Oligo...

UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 to hemimethylated DNA during replication and is essential for maintaining DNA methylation. uhrf1 mutant zebrafish have global DNA hypomethylation and display embryonic defects, including a small liver, and they die as larvae. We make the surprising finding that, despite their reduced organ size, uhrf1 mutants express high levels of genes controlling S-phase and have many more cells undergoing DNA replication, as measured by BrdU incorporation. In contrast to wild-type hepatocytes, which are continually dividing during hepatic outgrowth and thus dilute the BrdU label, uhrf1 mutant hepatocytes retain BrdU throughout outgrowth, reflecting cell cycle arrest. Pulse-chase-pulse experiments with BrdU and EdU, and DNA content analysis indicate that uhrf1 mutant cells undergo DNA re-replication and that apoptosis is the fate of many of the rereplicating and arrested hepatocytes. Importantly, the DNA rereplication phenotype and hepatic outgrowth failure are preceded by global loss of DNA methylation. Moreover, uhrf1 mutants are phenocopied by mutation of dnmt1, and Dnmt1 knockdown in uhrf1 mutants enhances their small liver phenotype. Together, these data indicate that unscheduled DNA replication and failed cell cycle progression leading to apoptosis are the mechanisms by which DNA hypomethylation prevents organ expansion in uhrf1 mutants. We propose that cell cycle arrest leading to apoptosis is a strategy that restricts propagation of epigenetically damaged cells during embryogenesis.

Complex cytoplasmic nucleotide-sensing mechanisms can recognize foreign DNA based on a lack of methylation and initiate an immune response to clear the infection. Zebrafish embryos with global DNA hypomethylation caused by mutations in the () or () genes exhibit a robust interferon induction characteristic of the first line of defense against viral infection. We found that this interferon induction occurred in non-immune cells and examined whether intracellular viral sensing pathways in these cells were the trigger. RNA-seq analysis of and mutants revealed widespread induction of Class I retrotransposons and activation of cytoplasmic DNA viral sensors. Attenuating Sting, phosphorylated Tbk1 and, importantly, blocking reverse transcriptase activity suppressed the expression of interferon genes in mutants. Thus, activation of transposons in cells with global DNA hypomethylation mimics a viral infection by activating cytoplasmic DNA sensors. This suggests that antiviral pathways serve as surveillance of cells that have derepressed intragenomic parasites due to DNA hypomethylation.