Profiled Regulatory Sites Useful for Gene Control
Introduction The invention provides methods for quantitative profiling of chromatin sensitivity to a DNA modifying agent. The invention also relates to methods for identifying regulatory sites in a genomic locus and to methods for determining chromatin architecture in a genomic locus. The invention also relates to the use of profiled regulatory sites, databases comprising the same, and their use in regulating gene expression, disease diagnosis and therapy, and identification of therapeutic drugs. Regulatory site profiles associated with specific genes are particularly useful for discovery of medicinal agents, other genomic sequences involved in gene regulation, and regulatory mechanisms that are involved in health and disease. The regulatory sequence profiles also are highly valuable when used by computer programs for comparing known and unknown genetic sequences by a large variety of experimental and computer manipulations. Technology description Understanding the human genome requires comprehensive identification of DNA elements that are functional in vivo. A major class of such sequences is that which has a role in regulating genomic activity. Regulatory factors interact with chromatin in a site-specific fashion to bring the genome to life. All genes are controlled at multiple levels through the interaction of regulatory factors with gene proximal or, in some cases, distant cis-regulatory sites. The nucleoprotein complexes formed by such interactions may be tissue or developmental stage-specific, or they may be constitutive, depending on the regulatory requirements of their cognate gene. The basic chromatin fiber consists of an array of nucleosomes, each packaging around 200 base pairs of DNA; 146 is wound around the histone octamer, with the remainder forming a link to the next nucleosome. In eukaryotic cells, all genomic DNA in the nucleus is packaged into chromatin, the architecture of which plays a central role in regulating gene expression Activation of tissue-specific genes during development and differentiation occurs first at the level of chromatin accessibility and results in the formation of transcriptionally-competent genetic loci characterized by increased sensitivity. Loci in an accessible chromatin configuration can subsequently respond to acutely activating signals, often conveyed by non-tissue-specific transcriptional factors that can gain access to the open locus and recruit or activate the basal transcriptional machinery. The invention provides a method for profiling chromatin sensitivity of a genomic region of cells of a cell type to digestion by a DNA modifying agent. The method comprises determining a chromatin sensitivity profile, said chromatin sensitivity profile comprising a plurality of replicate measurements of each of a plurality of different genomic sequences in said genomic region, wherein each of said plurality of replicate measurements is a ratio of (i) copy numbers of an amplicon comprising said genomic sequence measured by real-time quantitative PCR (qPCR) with chromatin of said cell type that has been treated with said DNA modifying agent and (ii) copy numbers of said amplicon measured by real-time PCR with chromatin of said cell type that has not been treated with said DNA modifying agent. In a preferred embodiment, the plurality of different genomic sequences comprises successively overlapping sequences tiled across one or more portions of said genomic region. In another preferred embodiment, the plurality of different genomic sequences comprises successively overlapping sequences tiled across said genomic region. In another embodiment, the invention provides a method for profiling chromatin sensitivity of a genomic region of cells of a cell type to digestion by a DNA modifying agent. In a preferred embodiment, copy numbers are corrected for difference in amplification efficiency. In another preferred embodiment, the DNA modifying agent is DNase I. The plurality of duplicated measurements can be measured by independent real-time PCR experiments. The plurality of duplicated measurements can also measured by independent real-timeqPCR experiments using different treated chromatin samples. The invention further provides methods of using regulatory sequences profiles for a variety of purposes related generally to gene regulation, cell characterization and identification of drugs and therapies. In one embodiment, the invention provides a method of identifying a gene associated with a disease or disorder, such as cancer. Another embodiment of the invention provides a method of detecting a disease or disorder in a subject, comprising identifying an regulatory sequence profile associated with a disease or disorder; determining an regulatory sequence profile of a subject; and comparing the regulatory sequence profile of the subject to the regulatory sequence profile associated with the disease or disorder. Other embodiments includes: a method of qualifying a patient for a clinical trial, provides a method of selecting a therapy for a patient, a method of screening a drug candidate, a method of identifying a drug useful in treating a disease or disorder, a method of regulating gene expression, regulatory sequences and allelic variants identified by using this invention. Business opportunity Fundamentally, the invention allows the identification and use of regulatory sequences (RSs)
associated with a gene. Such elements and their activities may be used for a variety of purposes, including the establishment of regulatory profiles (regulatory sequence profiles) associated with different cells. Such regulatory profiles may be used to characterize an entire gene system, including the coding and transcribed regions, as well as surrounding sequences that cooperatively regulate gene expression. This technology can be used in drug development, drug screening, diagnosis, and therapeutic fields. Intellectual property position European and U.S. patent applications are pending.
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