Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-22
2001-10-02
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007800, C435S029000, C536S023500
Reexamination Certificate
active
06297019
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The field of this invention is transcription factors which bind CYP7 promoters.
2. Background
In mammalian cells, cholesterol is an essential component for membranogenesis and for the synthesis of sterols and nonsterols that are critical for normal cellular functions. Excess cholesterol, however, not only is lethal to cells but also creates a major problem in atherolsclerosis for its deposit in arteries. To maintain cholesterol homeostasis, cells, in particular liver cells, adopt three major ways to regulate cholesterol levels: 1) uptake of dietary cholesterol via LDL receptor; 2) endogenous cholesterol biosynthesis and 3) metabolic conversion of cholesterol to bile acids. The key molecule that coordinates these processes is cholesterol itself, serving as a feedback signal. When the intracellular cholesterol level increases either through cholesterol uptake or biosynthesis, the transcription of genes including LDL receptor and the key cholesterol biosynthesis enzymes such as HMG-CoA synthase and HMG-CoA reductase is repressed. These feedback processes are mediated by a novel family of transcription factors called sterol regulatory element binding proteins (SREBPs). SREBPs contain an N-terminal transcription factor domain, two hydrophobic transmembrane domains and a C-terminal regulatory domain. When the intracellular cholesterol level is low, a two-step proteolytic cascade occurs which releases the N-terminal transcription factor domain of SREBPs from the endoplasmic reticulum, moving to the nucleus where activation of the SRE-containing genes occurs.
While the SREBP pathway is responsible for regulation of genes involved in cholesterol uptake and cholesterol biosynthesis such as LDL receptor and HMG-CoA synthase, the molecular basis of cholesterol catabolism is largely unknown. The major catabolic pathway for cholesterol removal is the production of bile acids that occurs exclusively in the liver. Cholesterol 7&agr;-hydroxylase is the first and rate-limiting enzyme in the pathway. The cholesterol 7&agr;-hydroxylase gene, also known as CYP7, belongs to the cytochrome P-450 family that contains many microsomal enzymes involved in liver metabolism. It has been shown that the expression of the CYP7 gene is tightly regulated: it is expressed exclusively in liver; its expression can be induced by dietary cholesterol and suppressed by bile acids. It has been shown that cholesterol catabolism plays a central role in cholesterol homeostasis. Treatment of laboratory animals with cholestid or cholestyramine, two bile acid-binding resins, decreases serum cholesterol levels. Moreover, overexpression of the CYP7 gene in hamsters reduces total and LDL cholesterol levels. Thus, cholesterol 7&agr;-hydroxylase is a potential therapeutic target for cholesterol lowering drugs and understanding the mechanisms by which expression of the CYP7 gene is regulated is of particular importance.
To study the molecular mechanisms of hepatic-specific expression of the human CYP7 gene, we used HepG2 cells as a model system since this cell line is one of the most studied hepatic cell lines and has been shown to be an appropriate cell line through studies of a number of hepatic-specific genes including the CYP7 gene. We started with DNase I hypersensitivity mapping of the human CYP7 promoter and identified a hepatic-specific element in the promoter. Consequently, we cloned the gene encoding the promoter-binding protein and identified it as a human ortholog of the nuclear orphan receptor Ftz-F1 family.
3. Relevant Art
Galarneau and Belanger (1997) unpublished, accession U93553, describe a human &agr;1-Fetoprotein Transcription Factor (hFTF, SEQ ID NOS:7 and 8); Tugwood, J. D. Issemann, I. and Green, S. (1991) unpublished, accession M81385, describe a mouse liver receptor homologous protein (LRH-1) mRNA and conceptual translate (mLRH, SEQ ID NOS:9 and 10); and L. Galarneau et al. (1996) Mol. Cell Biol. 16, 3853-3865 disclose a partial rat gene; all having sequence similarity to the disclosed CPF polypeptides.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to isolated CPF polypeptides, related nucleic acids, polypeptide domains thereof having CPF-specific structure and activity and modulators of CPF function, particularly CYP7 promoter binding. CPF polypeptides can regulate CYP7 promoter-linked gene activation and hence provide important regulators of cell function. The polypeptides may be produced recombinantly from transformed host cells from the subject CPF polypeptide encoding nucleic acids or purified from mammalian cells. The invention provides isolated CPF hybridization probes and primers capable of specifically hybridizing with the disclosed CPF gene, CPF-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for CPF transcripts), therapy (e.g. CPF activators to activate CYP7 promoter-dependent transcription) and in the biopharmaceutical industry (e.g. as immunogens, reagents for isolating other transcriptional regulators, reagents for screening chemical libraries for lead pharmacological agents, etc.).
DETAILED DESCRIPTION OF THE INVENTION
The nucleotide sequence of natural cDNAs encoding human CPF polypeptides are shown as SEQ ID NOS:1, 3 and 5, and the full conceptual translates are shown as SEQ ID NOS:2, 4 and 6, respectively. The CPF polypeptides of the invention include one or more functional domains of SEQ ID NO:2, 4 or 6, which domains comprise at least 8, preferably at least 16, more preferably at least 32, most preferably at least 64 contiguous residues of SEQ ID NO:2, 4 or 6 and have human CPF-specific amino acid sequence and activity. CPF domain specific activities include CYP7 promoter-binding or transactivation activity and CPF specific immunogenicity and/or antigenicity. CPF specific polypeptide sequences distinguish hFTF and mLRH (SEQ ID NOS:8 and 10), and are readily identified by sequence comparison; see, e.g. Tables 5, 6, and 7, herein. Exemplary sequences include 10 residue domains of SEQ ID NO:2 comprising at least one of residues 1-10, 11-15, 16-21, 204-207 and 299-307, 10 residue domains of SEQ ID NO:4 comprising residue 154, and 10 residue domains of SEQ ID NO:6 comprising at least one of residues 3-10, 13-22 and 30-38.
CPF-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, cell culture assays, in animals (e.g. gene therapy, transgenics, etc.), etc. Binding assays encompass any assay where the molecular interaction of an CPF polypeptide with a binding target is evaluated. The binding target may be a natural intracellular binding target such as a CYP7 promoter binding site, a CPF regulating protein or other regulator that directly modulates CPF activity or its localization; or non-natural binding target such as a specific immune protein such as an antibody, a synthetic nucleic acid binding site (see consensus sequences, below), or a CPF specific agent such as those identified in screening assays such as described below. CPF-binding specificity may be assayed by binding equilibrium constants (usually at least about 10
7
M
−1
, preferably at least about 10
8
M
−1
, more preferably at least about 10
9
M
−1
), by CYP7 or synthetic binding site reporter expression, by the ability of the subject polypeptides to function as negative mutants in CPF-expressing cells, to elicit CPF specific antibody in a heterologous host (e.g a rodent or rabbit), etc. For example, in this fashion, domains defined by SEQ ID NO:2, residues 33-123 are shown to provide a functional DNA binding domain, and those defined by SEQ ID NO:2, residues 242-333 and 383-405 are shown to provide a functional ligand binding domain.
In a particular embodiment, deletion mutagenesis is used to define functional CPF domains which bind CYP7 promoter elements (see Examples, below). See, e.g. Table 1.
TABLE 1
Exemplary CPF deletion mutants defining CPF fu
Nitta Masahiro
Shan Bei
McKelvey Terry
Osman Richard Aron
Tularik Inc.
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