CTCF binding contributes to the establishment of a higher-order genome structure by demarcating the boundaries of large-scale topologically associating domains (TADs). However, despite the importance ...and conservation of TADs, the role of CTCF binding in their evolution and stability remains elusive.
We carry out an experimental and computational study that exploits the natural genetic variation across five closely related species to assess how CTCF binding patterns stably fixed by evolution in each species contribute to the establishment and evolutionary dynamics of TAD boundaries. We perform CTCF ChIP-seq in multiple mouse species to create genome-wide binding profiles and associate them with TAD boundaries. Our analyses reveal that CTCF binding is maintained at TAD boundaries by a balance of selective constraints and dynamic evolutionary processes. Regardless of their conservation across species, CTCF binding sites at TAD boundaries are subject to stronger sequence and functional constraints compared to other CTCF sites. TAD boundaries frequently harbor dynamically evolving clusters containing both evolutionarily old and young CTCF sites as a result of the repeated acquisition of new species-specific sites close to conserved ones. The overwhelming majority of clustered CTCF sites colocalize with cohesin and are significantly closer to gene transcription start sites than nonclustered CTCF sites, suggesting that CTCF clusters particularly contribute to cohesin stabilization and transcriptional regulation.
Dynamic conservation of CTCF site clusters is an apparently important feature of CTCF binding evolution that is critical to the functional stability of a higher-order chromatin structure.
A large proportion of functional sequence within mammalian genomes falls outside protein-coding exons and can be transcribed into long RNAs. However, the roles in mammalian biology of long noncoding ...RNA (lncRNA) are not well understood. Few lncRNAs have experimentally determined roles, with some of these being lineage-specific. Determining the extent by which transcription of lncRNA loci is retained or lost across multiple evolutionary lineages is essential if we are to understand their contribution to mammalian biology and to lineage-specific traits. Here, we experimentally investigated the conservation of lncRNA expression among closely related rodent species, allowing the evolution of DNA sequence to be uncoupled from evolution of transcript expression. We generated total RNA (RNAseq) and H3K4me3-bound (ChIPseq) DNA data, and combined both to construct catalogues of transcripts expressed in the adult liver of Mus musculus domesticus (C57BL/6J), Mus musculus castaneus, and Rattus norvegicus. We estimated the rate of transcriptional turnover of lncRNAs and investigated the effects of their lineage-specific birth or death. LncRNA transcription showed considerably greater gain and loss during rodent evolution, compared with protein-coding genes. Nucleotide substitution rates were found to mirror the in vivo transcriptional conservation of intergenic lncRNAs between rodents: only the sequences of noncoding loci with conserved transcription were constrained. Finally, we found that lineage-specific intergenic lncRNAs appear to be associated with modestly elevated expression of genomically neighbouring protein-coding genes. Our findings show that nearly half of intergenic lncRNA loci have been gained or lost since the last common ancestor of mouse and rat, and they predict that such rapid transcriptional turnover contributes to the evolution of tissue- and lineage-specific gene expression.
Gene expression levels are thought to diverge primarily via regulatory mutations in trans within species, and in cis between species. To test this hypothesis in mammals we used RNA-sequencing to ...measure gene expression divergence between C57BL/6J and CAST/EiJ mouse strains and allele-specific expression in their F1 progeny. We identified 535 genes with parent-of-origin specific expression patterns, although few of these showed full allelic silencing. This suggests that the number of imprinted genes in a typical mouse somatic tissue is relatively small. In the set of nonimprinted genes, 32% showed evidence of divergent expression between the two strains. Of these, 2% could be attributed purely to variants acting in trans, while 43% were attributable only to variants acting in cis. The genes with expression divergence driven by changes in trans showed significantly higher sequence constraint than genes where the divergence was explained by variants acting in cis. The remaining genes with divergent patterns of expression (55%) were regulated by a combination of variants acting in cis and variants acting in trans. Intriguingly, the changes in expression induced by the cis and trans variants were in opposite directions more frequently than expected by chance, implying that compensatory regulation to stabilize gene expression levels is widespread. We propose that expression levels of genes regulated by this mechanism are fine-tuned by cis variants that arise following regulatory changes in trans, suggesting that many cis variants are not the primary targets of natural selection.
Molecular beacons are FRET-based target-activatable probes. They offer control of fluorescence emission in response to specific cancer targets, thus are useful tools for in vivo cancer imaging. ...Photodynamic therapy (PDT) is a cell-killing process by light activation of a photosensitizer (PS) in the presence of oxygen. The key cytotoxic agent is singlet oxygen (¹O₂). By combining these two principles (FRET and PDT), we have introduced a concept of photodynamic molecular beacons (PMB) for controlling the PS's ability to generate ¹O₂ and, ultimately, for controlling its PDT activity. The PMB comprises a disease-specific linker, a PS, and a ¹O₂ quencher, so that the PS's photoactivity is silenced until the linker interacts with a target molecule, such as a tumor-associated protease. Here, we report the full implementation of this concept by synthesizing a matrix metalloproteinase-7 (MMP7)-triggered PMB and achieving not only MMP7-triggered production of ¹O₂ in solution but also MMP7-mediated photodynamic cytotoxicity in cancer cells. Preliminary in vivo studies also reveal the MMP7-activated PDT efficacy of this PMB. This study validates the core principle of the PMB concept that selective PDT-induced cell death can be achieved by exerting precise control of the PS's ability to produce ¹O₂ by responding to specific cancer-associated biomarkers. Thus, PDT selectivity will no longer depend solely on how selectively the PS can be delivered to cancer cells. Rather, it will depend on how selective a biomarker is to cancer cells, and how selective the interaction of PMB is to this biomarker.
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.
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•Earliest steps of regulatory evolution in mammals captured using five mouse species•Interspecies differences in TF binding are rarely caused by DNA variation in motifs•Cobound TFs change their genomic binding cooperatively in closely related mammals•Genetic knockouts revealed the extent of cooperative stabilization in TF binding clusters
Microevolutionary mechanisms create different transcription factor binding patterns between mammals, shedding light on the regulatory mechanisms partially underlying speciation.
To understand the evolutionary dynamics between transcription factor (TF) binding and gene expression in mammals, we compared transcriptional output and the binding intensities for three ...tissue-specific TFs in livers from four closely related mouse species. For each transcription factor, TF-dependent genes and the TF binding sites most likely to influence mRNA expression were identified by comparing mRNA expression levels between wild-type and TF knockout mice. Independent evolution was observed genome-wide between the rate of change in TF binding and the rate of change in mRNA expression across taxa, with the exception of a small number of TF-dependent genes. We also found that binding intensities are preferentially conserved near genes whose expression is dependent on the TF, and the conservation is shared among binding peaks in close proximity to each other near the TSS. Expression of TF-dependent genes typically showed an increased sensitivity to changes in binding levels as measured by mRNA abundance. Taken together, these results highlight a significant tolerance to evolutionary changes in TF binding intensity in mammalian transcriptional networks and suggest that some TF-dependent genes may be largely regulated by a single TF across evolution.
As exome sequencing gives way to genome sequencing, the need to interpret the function of regulatory DNA becomes increasingly important. To test whether evolutionary conservation of cis-regulatory ...modules (CRMs) gives insight into human gene regulation, we determined transcription factor (TF) binding locations of four liver-essential TFs in liver tissue from human, macaque, mouse, rat, and dog. Approximately, two thirds of the TF-bound regions fell into CRMs. Less than half of the human CRMs were found as a CRM in the orthologous region of a second species. Shared CRMs were associated with liver pathways and disease loci identified by genome-wide association studies. Recurrent rare human disease causing mutations at the promoters of several blood coagulation and lipid metabolism genes were also identified within CRMs shared in multiple species. This suggests that multi-species analyses of experimentally determined combinatorial TF binding will help identify genomic regions critical for tissue-specific gene control.
We designed and synthesized a folate receptor-targeted, water-soluble, and pharmacomodulated photodynamic therapy (PDT) agent that selectively detects and destroys the targeted cancer cells while ...sparing normal tissue. This was achieved by minimizing the normal organ uptake (e.g., liver and spleen) and by discriminating between tumors with different levels of folate receptor (FR) expression. This construct (Pyro−peptide−Folate, PPF) is composed of three components: (1) pyropheophorbide a (Pyro) as an imaging and therapeutic agent, (2) peptide sequence as a stable linker and modulator improving the delivery efficiency, and (3) Folate as a homing molecule targeting FR-expressing cancer cells. We observed an enhanced accumulation of PPF in KB cancer cells (FR+) compared to HT 1080 cancer cells (FR−), resulting in a more effective post-PDT killing of KB cells over HT 1080 or normal CHO cells. The accumulation of PPF in KB cells can be up to 70% inhibited by an excess of free folic acid. The effect of Folate on preferential accumulation of PPF in KB tumors (KB vs HT 1080 tumors 2.5:1) was also confirmed in vivo. In contrast to that, no significant difference between the KB and HT 1080 tumor was observed in case of the untargeted probe (Pyro−peptide, PP), eliminating the potential influence of Pyro's own nonspecific affinity to cancer cells. More importantly, we found that incorporating a short peptide sequence considerably improved the delivery efficiency of the probea process we attributed to a possible peptide-based pharmacomodulationas was demonstrated by a 50-fold reduction in PPF accumulation in liver and spleen when compared to a peptide-lacking probe (Pyro−K−Folate, PKF). This approach could potentially be generalized to improve the delivery efficiency of other targeted molecular imaging and photodynamic therapy agents.
Molecular beacons are essentially all probes that illuminate particular cellular target or cells with similar characteristics. In this review we focus on those molecular beacons that use ...near-infrared fluorescence imaging (NIRF-I) to identify the unique cellular and metabolic markers characteristic of cancer. They employ various delivery and activation pathways, selectively or specifically targeting proliferating and immortal cancer cells. These beacons can either be used in an imaging step separate from therapy or they can intimately connect these two steps into a single process. Matching cancer therapy to NIRF-I is photodynamic therapy (PDT) that uses the light-triggered phototoxic properties of some porphyrin-based dyes. Guided by beacon's restored fluorescence, the PDT laser could be focused on affected sites, killing the cancer cells using the enhanced photoactivity of the same beacon. Or vice versa-the restored fluorescence from the cleaved beacon could be used as an indication of the beacon's own therapeutic success, imaging the post-PDT apoptotic cells.
Precisely localizing therapeutic agents in neoplastic areas would greatly improve their efficacy for killing tumor cells and reduce their toxicity to normal cells. Photodynamic therapy (PDT) is a ...promising cancer treatment modality, and near-infrared fluorescence imaging (NIRF-I) is a sensitive and noninvasive approach for in vivo cancer detection. This review focuses on the current efforts to engineer single molecule constructs that allow these two modalities to be combined to achieve a high level of selectivity for cancer treatment. The primary component of these so called killer beacons is a fluorescent photosensitizer responsible for both imaging and therapy. By attaching other components, e.g. various DNA- or peptide-based linkers, quenchers or cancer cell-specific delivery vehicles, their primary diagnostic and therapeutic functions as well as their target specificity and pharmacological properties can be modulated. This modular design makes these agents customizable, offering the ability to assemble a few simple and often interchangeable functional modules into beacons with totally different functions. This review will summarize following three types of killer beacons: photodynamic molecular beacons, traceable beacons and beacons with built-in apoptosis sensor. Despite the rapid progress in killer beacon development, numerous challenges remain before these beacons can be translated into clinics, such as photobleaching, delivery efficiency and cancerspecificity. In this review we outline the basic principles of killer beacons, the current achievements and future directions, including possible cancer targets and different therapeutic applications.
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