Combinatorial clustering of distinct DNA motifs directs synergistic binding of Caenorhabditis elegans dosage compensation complex to X chromosomes.

Organisms that count X-chromosome number to determine sex utilize dosage compensation mechanisms to balance X-gene expression between sexes. Typically, a regulatory complex is recruited to X chromosomes of one sex to modulate gene expression. A major challenge is to determine the mechanisms that target regulatory complexes specifically to X. Here, we identify critical X-sequence motifs in Caenorhabditis elegans that act synergistically in hermaphrodites to direct X-specific recruitment of the dosage compensation complex (DCC), a condensin complex. We find two DNA motifs that collaborate with a previously defined 12-bp motif called MEX (motif enriched on X) to mediate binding: MEX II, a 26-bp X-enriched motif and Motif C, a 9-bp motif that lacks X enrichment. Inserting both MEX and MEX II into a new location on X creates a DCC binding site equivalent to an endogenous recruitment site, but inserting only MEX or MEX II alone does not. Moreover, mutating MEX, MEX II, or Motif C in endogenous recruitment sites with multiple different motifs dramatically reduces DCC binding in vivo to nearly the same extent as mutating all motifs. Changing the orientation or spacing of motifs also reduces DCC binding. Hence, synergy in DCC binding via combinatorial clustering of motifs triggers DCC assembly specifically on X chromosomes. Using an in vitro DNA binding assay, we refine the features of motifs and flanking sequences that are critical for DCC binding. Our work reveals general principles by which regulatory complexes can be recruited across an entire chromosome to control its gene expression.

Novel allele-specific quantification methods reveal no effects of adult onset CAG repeats on HTT mRNA and protein levels.

Huntington's disease (HD) reflects dominant consequences of a CAG repeat expansion mutation in HTT. Expanded CAG repeat size is the primary determinant of age at onset and age at death in HD. Although HD pathogenesis is driven by the expanded CAG repeat, whether the mutation influences the expression levels of mRNA and protein from the disease allele is not clear due to the lack of sensitive allele-specific quantification methods and the presence of confounding factors. To determine the impact of CAG expansion at the molecular level, we have developed novel allele-specific HTT mRNA and protein quantification methods based on principles of multiplex ligation-dependent probe amplification and targeted MS/MS parallel reaction monitoring, respectively. These assays, exhibiting high levels of specificity and sensitivity, were designed to distinguish allelic products based upon expressed polymorphic variants in HTT, including rs149 109 767. To control for other cis-haplotype variations, we applied allele-specific quantification assays to a panel of HD lymphoblastoid cell lines, each carrying the major European disease haplotype (i.e. hap.01) on the mutant chromosome. We found that steady state levels of HTT mRNA and protein were not associated with expanded CAG repeat length. Rather, the products of mutant and normal alleles, both mRNA and protein, were balanced, thereby arguing that a cis-regulatory effect of the expanded CAG repeat is not a critical component of the underlying mechanism of HD. These robust allele-specific assays could prove valuable for monitoring the impact of allele-specific gene silencing strategies currently being explored as therapeutic interventions in HD.

The HTT CAG-Expansion Mutation Determines Age at Death but Not Disease Duration in Huntington Disease.

Huntington disease (HD) is caused by an expanded HTT CAG repeat that leads in a length-dependent, completely dominant manner to onset of a characteristic movement disorder. HD also displays early mortality, so we tested whether the expanded CAG repeat exerts a dominant influence on age at death and on the duration of clinical disease. We found that, as with clinical onset, HD age at death is determined by expanded CAG-repeat length and has no contribution from the normal CAG allele. Surprisingly, disease duration is independent of the mutation's length. It is also unaffected by a strong genetic modifier of HD motor onset. These findings suggest two parsimonious alternatives. (1) HD pathogenesis is driven by mutant huntingtin, but before or near motor onset, sufficient CAG-driven damage occurs to permit CAG-independent processes and then lead to eventual death. In this scenario, some pathological changes and their clinical correlates could still worsen in a CAG-driven manner after disease onset, but these CAG-related progressive changes do not themselves determine duration. Alternatively, (2) HD pathogenesis is driven by mutant huntingtin acting in a CAG-dependent manner with different time courses in multiple cell types, and the cellular targets that lead to motor onset and death are different and independent. In this scenario, processes driven by HTT CAG length lead directly to death but not via the striatal pathology associated with motor manifestations. Each scenario has important ramifications for the design and testing of potential therapeutics, especially those aimed at preventing or delaying characteristic motor manifestations.

Sequence-Level Analysis of the Major European Huntington Disease Haplotype.

Huntington disease (HD) reflects the dominant consequences of a CAG-repeat expansion in HTT. Analysis of common SNP-based haplotypes has revealed that most European HD subjects have distinguishable HTT haplotypes on their normal and disease chromosomes and that ∼50% of the latter share the same major HD haplotype. We reasoned that sequence-level investigation of this founder haplotype could provide significant insights into the history of HD and valuable information for gene-targeting approaches. Consequently, we performed whole-genome sequencing of HD and control subjects from four independent families in whom the major European HD haplotype segregates with the disease. Analysis of the full-sequence-based HTT haplotype indicated that these four families share a common ancestor sufficiently distant to have permitted the accumulation of family-specific variants. Confirmation of new CAG-expansion mutations on this haplotype suggests that unlike most founders of human disease, the common ancestor of HD-affected families with the major haplotype most likely did not have HD. Further, availability of the full sequence data validated the use of SNP imputation to predict the optimal variants for capturing heterozygosity in personalized allele-specific gene-silencing approaches. As few as ten SNPs are capable of revealing heterozygosity in more than 97% of European HD subjects. Extension of allele-specific silencing strategies to the few remaining homozygous individuals is likely to be achievable through additional known SNPs and discovery of private variants by complete sequencing of HTT. These data suggest that the current development of gene-based targeting for HD could be extended to personalized allele-specific approaches in essentially all HD individuals of European ancestry.

Impact of Neonatal Activation of Nuclear Receptor CAR (Nr1i3) on Cyp2 Gene Expression in Adult Mouse Liver.

Perinatal exposure to environmental chemicals is proposed to reprogram development and alter disease susceptibility later in life. Supporting this, neonatal activation of the nuclear receptor constitutive androstane receptor (CAR) (Nr1i3) by TCPOBOP was previously reported to induce persistent expression of mouse hepatic Cyp2 genes into adulthood, and was attributed to long-term epigenetic memory of the early life exposure. Here, we confirm that the same high-dose neonatal TCPOBOP exposure studied previously (3 mg/kg, 15x ED50) does indeed induce prolonged (12 weeks) increases in hepatic Cyp2 expression; however, we show that the persistence of expression can be fully explained by the persistence of residual TCPOBOP in liver tissue. When the long-term presence of TCPOBOP in tissue was eliminated by decreasing the neonatal TCPOBOP dose 22-fold (0.67× ED50), strong neonatal increases in hepatic Cyp2 expression were still obtained but did not persist into adulthood. Furthermore, the neonatal ED50-range TCPOBOP exposure did not sensitize mice to a subsequent, low-dose TCPOBOP treatment. In contrast, neonatal treatment with phenobarbital, a short half-life (t1/2 = 8 h) agonist of CAR and PXR (Nr1i2), induced high-level neonatal activation of Cyp2 genes and also altered their responsiveness to low-dose phenobarbital exposure at adulthood by either increasing (Cyp2b10) or decreasing (Cyp2c55) expression. Thus, neonatal xenobiotic exposure can reprogram hepatic Cyp2 genes and alter their responsiveness to exposures later in life. These findings highlight the need to carefully consider xenobiotic dose, half-life, and persistence in tissue when evaluating the long-term effects of early life environmental chemical exposures.

Haplotype-specific insertion-deletion variations for allele-specific targeting in Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expanded CAG repeat in huntingtin (HTT). Given an important role for HTT in development and significant neurodegeneration at the time of clinical manifestation in HD, early treatment of allele-specific drugs represents a promising strategy. The feasibility of an allele-specific antisense oligonucleotide (ASO) targeting single-nucleotide polymorphisms (SNPs) has been demonstrated in models of HD. Here, we constructed a map of haplotype-specific insertion-deletion variations (indels) to develop alternative mutant-HTT-specific strategies. We mapped indels annotated in the 1000 Genomes Project data on common HTT haplotypes, revealing candidate indels for mutant-specific HTT targeting. Subsequent sequencing of an HD family confirmed candidate sites and revealed additional allele-specific indels. Interestingly, the most common normal HTT haplotype carries indels of big allele length differences at many sites, further uncovering promising haplotype-specific targets. When patient-derived cells carrying the most common HTT diplotype were treated with ASOs targeting the mutant alleles of candidate indels (rs772629195 or rs72239206), complete mutant specificity was observed. In summary, our map of haplotype-specific indels permits the identification of allele-specific targets in HD subjects, potentially contributing to the development of safe HTT-lowering therapeutics that are suitable for early treatment in HD.

Widespread Epigenetic Changes to the Enhancer Landscape of Mouse Liver Induced by a Specific Xenobiotic Agonist Ligand of the Nuclear Receptor CAR.

Constitutive androstane receptor (CAR) (Nr1i3), a liver nuclear receptor and xenobiotic sensor, induces drug, steroid, and lipid metabolism and dysregulates genes linked to hepatocellular carcinogenesis, but its impact on the liver epigenome is poorly understood. TCPOBOP (1, 4-bis-[2-(3, 5-dichloropyridyloxy)]benzene), a halogenated xenochemical and highly specific CAR agonist ligand, induces localized chromatin opening or closing at several thousand mouse liver genomic regions, discovered as differential DNase-hypersensitive sites (ΔDHS). Active enhancer and promoter histone marks induced by TCPOBOP were enriched at opening DHS and TCPOBOP-inducible genes. Enrichment of CAR binding and CAR motifs was seen at opening DHS and their inducible drug/lipid metabolism gene targets, and at many constitutively open DHS located nearby. TCPOBOP-responsive cell cycle and DNA replication genes codependent on MET/EGFR signaling for induction were also enriched for CAR binding. A subset of opening DHS and many closing DHS mapping to TCPOBOP-responsive target genes did not bind CAR, indicating an indirect mechanism for their changes in chromatin accessibility. TCPOBOP-responsive DHS were also enriched for induced binding of RXRA, CEBPA, and CEBPB, and for motifs for liver-enriched factors that may contribute to liver-specific transcriptional responses to TCPOBOP exposure. These studies elucidate the enhancer landscape of TCPOBOP-exposed liver and the widespread epigenetic changes that are induced by both direct and indirect mechanisms linked to CAR activation. The global maps of thousands of environmental chemical-induced epigenetic changes described here constitute a rich resource for further research on xenochemical effects on liver chromatin states and the epigenome.

X Chromosome Domain Architecture Regulates Caenorhabditis elegans Lifespan but Not Dosage Compensation.

Mechanisms establishing higher-order chromosome structures and their roles in gene regulation are elusive. We analyzed chromosome architecture during nematode X chromosome dosage compensation, which represses transcription via a dosage-compensation condensin complex (DCC) that binds hermaphrodite Xs and establishes megabase-sized topologically associating domains (TADs). We show that DCC binding at high-occupancy sites (rex sites) defines eight TAD boundaries. Single rex deletions disrupted boundaries, and single insertions created new boundaries, demonstrating that a rex site is necessary and sufficient to define DCC-dependent boundary locations. Deleting eight rex sites (8rexΔ) recapitulated TAD structure of DCC mutants, permitting analysis when chromosome-wide domain architecture was disrupted but most DCC binding remained. 8rexΔ animals exhibited no changes in X expression and lacked dosage-compensation mutant phenotypes. Hence, TAD boundaries are neither the cause nor the consequence of DCC-mediated gene repression. Abrogating TAD structure did, however, reduce thermotolerance, accelerate aging, and shorten lifespan, implicating chromosome architecture in stress responses and aging.

HD CAGnome: a search tool for huntingtin CAG repeat length-correlated genes.

BACKGROUND: The length of the huntingtin (HTT) CAG repeat is strongly correlated with both age at onset of Huntington's disease (HD) symptoms and age at death of HD patients. Dichotomous analysis comparing HD to controls is widely used to study the effects of HTT CAG repeat expansion. However, a potentially more powerful approach is a continuous analysis strategy that takes advantage of all of the different CAG lengths, to capture effects that are expected to be critical to HD pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: We used continuous and dichotomous approaches to analyze microarray gene expression data from 107 human control and HD lymphoblastoid cell lines. Of all probes found to be significant in a continuous analysis by CAG length, only 21.4% were so identified by a dichotomous comparison of HD versus controls. Moreover, of probes significant by dichotomous analysis, only 33.2% were also significant in the continuous analysis. Simulations revealed that the dichotomous approach would require substantially more than 107 samples to either detect 80% of the CAG-length correlated changes revealed by continuous analysis or to reduce the rate of significant differences that are not CAG length-correlated to 20% (n = 133 or n = 206, respectively). Given the superior power of the continuous approach, we calculated the correlation structure between HTT CAG repeat lengths and gene expression levels and created a freely available searchable website, "HD CAGnome," that allows users to examine continuous relationships between HTT CAG and expression levels of ∼20,000 human genes. CONCLUSIONS/SIGNIFICANCE: Our results reveal limitations of dichotomous approaches compared to the power of continuous analysis to study a disease where human genotype-phenotype relationships strongly support a role for a continuum of CAG length-dependent changes. The compendium of HTT CAG length-gene expression level relationships found at the HD CAGnome now provides convenient routes for discovery of candidates influenced by the HD mutation.

Esculetin suppresses proliferation of human colon cancer cells by directly targeting ß-catenin.

The Wnt pathway is a promising therapeutic and preventive target in various human cancers. The transcriptional complex of ß-catenin-T-cell factor (Tcf), a key mediator of canonical Wnt signaling, has been implicated in human colon cancer development. Current treatment of colon cancer depends on traditional cytotoxic agents with limited effects. Therefore, the identification of natural compounds that can disrupt the ß-catenin-TcF complex to suppress cancer cell growth with fewer adverse side effects is needed. To identify compounds that inhibit the association between ß-catenin and Tcf, we used computer docking to screen a natural compound library. Esculetin, also known as 6,7-dihydroxycoumarin, is a derivative of coumarin and was identified as a potential small-molecule inhibitor of the Wnt-ß-catenin pathway. We then evaluated the effect of esculetin on the growth of various human colon cancer cell lines and its effect on Wnt-ß-catenin signaling in cells and in an embryonic model. Esculetin disrupted the formation of the ß-catenin-Tcf complex through direct binding with the Lys312, Gly307, Lys345, and Asn387 residues of ß-catenin in colon cancer cells. In addition, esculetin effectively decreased viability and inhibited anchorage-independent growth of colon cancer cells. Esculetin potently antagonized the cellular effects of ß-catenin-dependent activity, and in vivo treatment with esculetin suppressed tumor growth in a colon cancer xenograft mouse model. Our data indicate that the interaction between esculetin and ß-catenin inhibits the formation of the ß-catenin-Tcf complex, which could contribute to esculetin's positive therapeutic and preventive effects against colon carcinogenesis.

Application of hypoiodite-mediated aminyl radical cyclization to synthesis of solasodine acetate.

Solasodine acetate, an anticancer steroidal alkaloid, was synthesized from diosgenin in 8 steps with an overall yield of 23%. A key synthetic step involves the formation of 5/6-oxazaspiroketal moiety via hypoiodite-mediated aminyl radical cyclization of a steroidal primary amine.