Transcriptional regulators and methods thereof
AssigneeWhitehead Institute for Biomedical Research
InventorsDuncan Odom, Richard Young
Life of this patent
Estimating the term…
Abstract
The invention relates to transcriptional regulators and related methods thereof. The invention further relates to the identification of genes regulated by transcriptional regulators, to the treatment of diseases associated with abnormal function of a transcriptional regulator and to the modulation of gene expression, including genes expressed in hepatocytes or pancreatic cells, through the modulation of transcriptional regulator activity.
Claims35
A method of determining which genes from a subset of genes are regulated by a transcriptional regulator expressed in a cell, the method comprising
- (a) selectively isolating chromatin from the cell to generate isolated chromatin;
- (b) selectively isolating chromatin fragments from the isolated chromatin to generate bound chromatin fragments, wherein the bound chromatin fragments are bound by the transcriptional regulator;
- (c) amplifying both the bound chromatin fragments to generate amplified chromatin fragments and the isolated chromatin to generate amplified control chromatin;
- (d) hybridizing the amplified control chromatin and the amplified chromatin fragments to a DNA microarray, wherein the DNA microarray comprises
- (1) at least 10,000 experimental spots, each experimental spot comprising an experimental DNA, each experimental DNA comprising a promoter region from a gene in the subset;
- and
- (2) at least 100 control spots, each control spot comprising a control DNA, each control DNA comprising a non-promoter region;
- and
- (e) determining and comparing a hybridization signal at each of the spots on the microarray between those generated by
- (1) the amplified control chromatin;
- and
- (2) the amplified chromatin fragments;
- wherein a gene in the subset is said to be regulated by the transcriptional regulator in the cell if a spot comprising a promoter region of said gene displays a higher level of hybridization by the amplified chromatin fragments than by the amplified control chromatin.
5-11. (canceled)
15-17. (canceled)
A DNA microarray for determining promoter occupancy in a human cell, the microarray comprising
- (1) at least 10,000 experimental spots, each experimental spot comprising an experimental DNA, each experimental DNA comprising a promoter region from a human gene in the subset;
- and
- (2) at least 100 control spots, each control spot comprising a control DNA, each control DNA comprising a non-promoter region;
- wherein at least 75% of the promoter regions comprise from at least 700 bp upstream to at least 200 bp downstream of the transcriptional start site.
A method of estimating if a transcriptional regulator is a global transcriptional regulator, the method comprising
- (a) selectively isolating chromatin from a tissue;
- (b) identifying promoter regions from the chromatin which are bound by a candidate global transcriptional regulator;
- (c) identifying promoter regions from the chromatin which are bound by a member of the basal transcriptional machinery;
- and
- (d) comparing the promoter regions identified in steps (b) and (c) to determine the ratio between (i) the number of promoter regions bound by both the candidate global transcriptional regulator and the member of the basal transcriptional machinery;
- and (ii) the number of promoter regions bound by the member of the basal transcriptional machinery
- wherein a transcriptional regulator is a global transcriptional regulator when the ratio is greater than 0.2.
27-31. (canceled)
A method of identifying at least one target gene for the development of a therapeutic to treat or prevent a disorder in a subject, wherein at least one form of the disorder is caused by an altered activity in a transcriptional regulator or in a suspected transcriptional regulator, the method comprising
- (a) identifying the genes regulated by the transcriptional regulator in a cell;
- (b) determining if the transcriptional regulator is a broad-acting transcriptional regulator or a narrow-acting transcriptional regulator, wherein if the transcriptional regulator is a broad acting transcriptional regulator then the transcriptional regulator is a target gene for the development of a therapeutic, and wherein if the transcriptional regulator is a narrow acting transcriptional regulator then
- (i) determining if at least one gene regulated by the transcriptional regulator is likely causative in the disorder, wherein a gene that is likely causative in the disorder is a target gene for the development of a therapeutic;
- and
- (ii) reiterating steps (a) and (b) for at least one gene that is regulated by the transcriptional regulator in the cell and that either
- (1) encodes a transcriptional regulator or
- (2) is suspected to encode a transcriptional regulator,
- with the modification that the transcriptional regulator of steps (a) and (b) is said gene,
- thereby identifying at least one target gene for the development of a therapeutic to treat or prevent a disorder in the subject.
35-38. (canceled)
46-53. (canceled)
A method of treating or preventing type II diabetes in a subject, comprising administering to the subject a therapeutically effective amount of an agent that increases the global transcriptional activity of HNF4alpha.
A method of treating or preventing a disorder associated with low transcriptional activity of HNF4alpha in a subject, comprising administering to the subject a therapeutically effective amount of an agent that increases the global transcriptional activity of HNF4alpha.
56-64. (canceled)
A method of identifying transcriptionally active genes that are regulated by a transcriptional regulator in a cell, the method comprising
- (a) selectively isolating chromatin from a tissue;
- (b) identifying promoter regions from the chromatin that are bound by the transcriptional regulator;
- (c) identifying promoter regions from the chromatin that are bound by a member of the basal transcriptional machinery;
- and
- (d) comparing the promoter regions identified in steps (b) and (c) to determine overlapping genes,
- wherein the overlapping genes are transcriptionally active genes regulated by the transcriptional regulator.
Dependencies are inferred from the claim text.