Methods for designing exogenous regulatory molecules

• US 6,511,808 B2
• Filed: 04/27/2001
• Issued: 01/28/2003
• Est. Priority Date: 04/28/2000
• Status: Expired due to Term

• Current Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Collingwood, Trevor, Johnstone, Brian, Wolffe, Alan, Guschin, Dmitry, Li, Xiao-Yong, Urnov, Fyodor
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/844,493
• Publication Number:
  US 20020076711A1
• Time in Patent Office: 641 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 436/94
• US Class Current: 435/6.12
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/24 containing a MBP (maltose binding protein)-tag
• CPC Class Codes: C07K 2319/43 containing a FLAG-tag
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 50/00 ICT programming tools or database systems specially adapted for bioinformatics

Abstract

Methods for the design of exogenous regulatory molecules, comprising a DNA-binding domain and a functional domain, are provided. The methods rely on the identification, isolation and characterization of regulatory DNA sequences obtained on the basis of their accessibility in cellular chromatin.

Claims

1. A method for designing one or more exogenous regulatory molecules for regulating a gene of interest, the method comprising:

(a) providing one or more collections of polynucleotide sequences, each collection comprising a plurality of polynucleotide sequences corresponding to accessible regions of cellular chromatin in a sample;

(b) identifying one or more sequence elements from the collection, wherein the one or more sequence elements are potential regulatory sequences for the gene of interest; and

(c) preparing an exogenous regulatory molecule that comprises a DNA binding domain and a functional domain that activates or represses transcription of the gene of interest, wherein said preparing comprises selecting the DNA binding domain, the functional domain or both the DNA binding domain and the functional domain based upon the identified sequence elements.

2. The method of claim 1, wherein each of the collections is stored on a computer-readable medium and identifying is performed with a computer.

3. The method of claim 1, wherein the collection of polynucleotide sequences is obtained by a method that comprises:

(a) treating cellular chromatin with a chemical or enzymatic probe wherein the probe reacts with accessible polynucleotide sequences;

(b) fragmenting the treated chromatin to produce a collection of polynucleotide fragments, wherein the collection comprises marked polynucleotides and unmarked polynucleotides, and wherein each marked polynucleotide contains one or more sites of probe reaction;

(c) collecting marked polynucleotides, wherein the marked polynucleotides comprise polynucleotide sequences present in accessible regions of cellular chromatin; and

(d) determining the nucleotide sequences of the marked polynucleotides to obtain the collection of polynucleotide sequences.

4. The method of claim 1, wherein the collection of polynucleotide sequences is obtained by a method that comprises:

(a) treating cellular chromatin with a methylase to generate methylated chromatin;

(b) deproteinizing the methylated chromatin to form deproteinized chromatin;

(c) digesting the deproteinized chromatin with a methylation-dependent restriction enzyme to produce a collection of restriction fragments, wherein the collection comprises methylated polynucleotides and non-methylated polynucleotides;

(d) collecting non-methylated polynucleotides, wherein the termini of the non-methylated polynucleotides correspond to accessible regions of cellular chromatin; and

(e) determining the nucleotide sequences of the termini of the non-methylated polynucleotides to obtain the collection of polynucleotide sequences.

5. The method of claim 1, wherein the collection of polynucleotide sequences is obtained by a method that comprises:

(a) treating cellular chromatin with a methylase to generate methylated chromatin;

(b) deproteinizing the methylated chromatin to form deproteinized chromatin;

(c) digesting the deproteinized chromatin with a methylation-dependent restriction enzyme to produce a collection of restriction fragments, wherein the collection comprises methylated polynucleotides and non-methylated polynucleotides;

(d) collecting methylated polynucleotides, wherein the methylated polynucleotides correspond to accessible regions of cellular chromatin; and

(e) determining the nucleotide sequences of the methylated polynucleotides to obtain the collection of polynucleotide sequences.

6. The method of claim 1, wherein the collection of polynucleotides is obtained by a method that comprises:

(a) treating cellular chromatin with a nuclease;

(b) collecting polynucleotide fragments released by nuclease treatment, wherein the released polynucleotide fragments are derived from accessible regions of cellular chromatin; and

(c) determining the nucleotide sequences of the released polynucleotide fragments to obtain the collection of polynucleotide sequences.

7. The method of claim 1, wherein the collection of polynucleotides is obtained by a method that comprises:

(a) treating cellular chromatin with a methylation-sensitive enzyme that cleaves at unmethylated CpG sequences;

(b) collecting short polynucleotide fragments released by enzyme treatment;

wherein the polynucleotide fragments are derived from regulatory regions of cellular chromatin; and

(c) determining the nucleotide sequences of the released polynucleotide fragments to obtain the collection of polynucleotide sequences.

8. The method of claim 1, wherein the collection of polynucleotides is obtained by a method that comprises:

(a) treating cellular DNA with an agent that selectively cleaves AT-rich sequences;

(b) collecting large polynucleotide fragments released by the treatment;

wherein the large polynucleotide fragments comprise regulatory regions; and

(c) determining the nucleotide sequences of the large polynucleotide fragments to obtain the collection of polynucleotide sequences.

9. The method of claim 1, wherein the collection of polynucleotides is obtained by a method that comprises:

(a) treating cellular DNA with an agent that selectively cleaves AT-rich sequences to form a mixture of methylated and unmethylated fragments enriched in CpG islands;

(b) separating the unmethylated fragments from the methylated fragments to obtain a collection of unmethylated fragments enriched in CpG islands, wherein the unmethylated fragments are derived from regulatory regions of cellular chromatin; and

(c) determining the nucleotide sequences of the unmethylated fragments to obtain the collection of polynucleotide sequences.

10. The method of claim 1, wherein the collection of polynucleotides is obtained by a method that comprises:

(a) fragmenting chromatin;

(b) contacting the fragments with an antibody that specifically binds to acetylated histones, thereby forming an immunoprecipitate enriched in polynucleotides corresponding to accessible regions;

(c) collecting the polynucleotides from the immunoprecipitate; and

(d) determining the nucleotide sequences of the collected polynucleotides to obtain the collection of polynucleotide sequences.

11. The method of claim 1, wherein the collection of polynucleotides is obtained by a method that comprises:

(a) reacting cellular chromatin with a chemical or enzymatic probe to generate chromatin-associated DNA fragments, wherein the DNA fragments comprise, at their termini, sites of probe reaction which identify accessible regions of cellular chromatin;

(b) attaching an adapter polynucleotide to the termini generated by the probe to generate adapter-ligated fragments; and

(c) amplifying the adapter-ligated fragments in the presence of a first primer that is complementary to the adapter and a second primer that is complementary to a segment of a gene of interest to form one or more amplified products; and

(d) determining the nucleotide sequences of the amplified products to obtain the collection of polynucleotide sequences.

12. The method of claim 11 wherein a plurality of second primers, each complementary to a segment of a different gene of interest, are used, to generate a plurality of amplification products.

13. The method of claim 1, wherein the collection of polynucleotides is obtained by a method that comprises:

(a) reacting cellular chromatin with a chemical or enzymatic probe to generate chromatin-associated DNA fragments, wherein the DNA fragments comprise, at their termini, sites of probe reaction which identify accessible regions of cellular chromatin;

(b) attaching a first adapter polynucleotide to the termini generated by the probe to generate adapter-ligated fragments;

(c) digesting the adapter-ligated fragments with a restriction enzyme to generate a population of digested fragments, wherein the population comprises digested fragments having a first end that comprises the first adapter and a second end formed via the activity of the restriction enzyme;

(d) contacting the digested fragments with a primer complementary to the first adapter under conditions wherein the primer is extended to generate a plurality of extension products, each comprising a first end that comprises the first adapter and a second end that can be attached to a second adapter polynucleotide;

(e) joining the second adapter to the second end of each of the plurality of extension products to form a plurality of modified fragments, each of which comprises the first and second adapters at its first and second end, respectively;

(f) amplifying the plurality of modified fragments in the presence of primers complementary to the sequences of the first and second adapters to generate a population of amplified products comprising sequences corresponding to accessible regions of cellular chromatin; and

determining the nucleotide sequences of the amplified products to obtain the collection of polynucleotide sequences.

14. The method of claim 1, wherein the identifying comprises identifying a functional accessible region and determining whether the functional accessible region comprises a binding site for a transcription factor;

and the selecting comprises choosing the functional domain of the exogenous regulatory molecule to be the same as the functional domain of the transcription factor.

15. The method of claim 1, wherein the identifying comprises identifying a gateway accessible region;

and the selecting comprises choosing the DNA binding domain of the exogenous regulatory molecule to specifically bind to a segment of the gateway accessible region.

16. The method of claim 15, wherein the DNA binding domain is a zinc finger DNA-binding domain.

17. The method of claim 1, wherein the identifying comprises identifying a gateway accessible region and determining whether the gateway accessible region comprises a binding site for a known transcription factor;

and the selecting comprises choosing the DNA binding domain of the exogenous regulatory molecule to be the same as the DNA binding domain of the known transcription factor.

18. A method for designing one or more exogenous regulatory molecules for regulating a gene of interest, the method comprising:

(a) providing one or more polynucleotide sequences corresponding to one or more accessible regions of cellular chromatin, wherein the accessible regions are related to the gene of interest;

(b) identifying one or more potential regulatory sequence elements from the polynucleotide sequences; and

(c) preparing an exogenous regulatory molecule that comprises a DNA binding domain and a functional domain that activates or represses the gene of interest, wherein said preparing comprises selecting the DNA binding domain, the functional domain or both the DNA binding domain and the functional domain based upon the identified regulatory sequences.

19. The method of claim 18, wherein the polynucleotide sequences are obtained by a method comprising:

(a) treating cellular chromatin related to the gene of interest with a chemical or enzymatic probe wherein the probe reacts with accessible polynucleotide sequences;

(b) fragmenting the treated chromatin to produce polynucleotide fragments comprising marked polynucleotides and unmarked polynucleotides, and wherein each marked polynucleotide contains one or more sites of probe reaction;

(c) collecting the marked polynucleotides, wherein the marked polynucleotides comprise polynucleotide sequences present in one or more accessible regions related to the gene of interest; and

(d) determining the nucleotide sequences of the marked polynucleotides to obtain the potential regulatory sequences.

20. The method of claim 18, wherein the polynucleotide sequences are obtained by a method comprising:

(a) treating cellular chromatin related to the gene of interest with a methylase to generate methylated chromatin;

(b) deproteinizing the methylated chromatin to form deproteinized chromatin;

(c) digesting the deproteinized chromatin with a methylation-dependent restriction enzyme to produce a plurality of restriction fragments, wherein the restriction fragments comprise methylated polynucleotides and non-methylated polynucleotides;

(d) collecting non-methylated polynucleotides, wherein the termini of the non-methylated polynucleotides correspond to accessible regions of cellular chromatin related to the gene of interest; and

(e) determining the nucleotide sequences of the termini of the non-methylated polynucleotides to obtain the polynucleotide sequences.

21. The method of claim 18, wherein the polynucleotide sequences are obtained by a method comprising:

(a) treating cellular chromatin related to the gene of interest with a methylase to generate methylated chromatin;

(b) deproteinizing the methylated chromatin to form deproteinized chromatin;

(c) digesting the deproteinized chromatin with a methylation-dependent restriction enzyme to produce a plurality of restriction fragments, wherein the restriction fragments comprise methylated polynucleotides and non-methylated polynucleotides;

(d) collecting methylated polynucleotides, wherein the methylated polynucleotides correspond to accessible regions related to the gene of interest; and

(e) determining the nucleotide sequences of the methylated polynucleotides to obtain the polynucleotide sequences.

22. The method of claim 18, wherein the polynucleotides are obtained by a method comprising:

(a) treating cellular chromatin related to the gene of interest with a nuclease;

(b) collecting polynucleotide fragments released by nuclease treatment, wherein the released polynucleotide fragments are derived from accessible regions related to the gene of interest; and

(c) determining the nucleotide sequences of the released polynucleotide fragments to obtain the polynucleotide sequences.

23. The method of claim 18, wherein the polynucleotides are obtained by a method comprising:

(a) treating cellular chromatin related to the gene of interest with a methylation-sensitive enzyme that cleaves at unmethylated CpG sequences;

(b) collecting short polynucleotide fragments released by enzyme treatment;

wherein the polynucleotide fragments are derived from regulatory regions of the gene of interest; and

(c) determining the nucleotide sequences of the released polynucleotide fragments to obtain the polynucleotide sequences.

24. The method of claim 18, wherein the polynucleotides are obtained by a method comprising:

(a) treating cellular DNA related to the gene of interest with an agent that selectively cleaves AT-rich sequences;

(b) collecting large polynucleotide fragments released by the treatment;

wherein the large polynucleotide fragments comprise regulatory regions; and

(c) determining the nucleotide sequences of the large polynucleotide fragments to obtain the polynucleotide sequences.

25. The method of claim 18, wherein the polynucleotides are obtained by a method comprising:

(a) treating cellular DNA related to the gene of interest with an agent that selectively cleaves AT-rich sequences to form a mixture of methylated and unmethylated fragments enriched in CpG islands;

(b) separating the unmethylated fragments from the methylated fragments to obtain a collection of unmethylated fragments enriched in CpG islands, wherein the unmethylated fragments are derived from regulatory regions of the gene of interest; and

(c) determining the nucleotide sequences of the unmethylated fragments to obtain the polynucleotide sequences.

26. The method of claim 18, wherein the polynucleotides are obtained by a method comprising:

(a) fragmenting chromatin related to the gene of interest;

(b) contacting the fragments with an antibody that specifically binds to acetylated histones, thereby forming an immunoprecipitate enriched in polynucleotides corresponding to accessible regions;

(c) collecting the polynucleotides from the immunoprecipitate; and

(d) determining the nucleotide sequences of the collected polynucleotides to obtain the polynucleotide sequences.

27. The method of claim 18, wherein the polynucleotides are obtained by a method comprising:

(a) reacting cellular chromatin related to the gene of interest with a chemical or enzymatic probe to generate chromatin-associated DNA fragments, wherein the DNA fragments comprise, at their termini, sites of probe reaction which identify accessible regions of cellular chromatin;

(b) attaching an adapter polynucleotide to the termini generated by the probe to generate adapter-ligated fragments; and

(c) amplifying the adapter-ligated fragments in the presence of a first primer that is complementary to the adapter and a second primer that is complementary to a segment of a gene of interest to form one or more amplified products; and

(d) determining the nucleotide sequences of the amplified products to obtain the polynucleotide sequences.

28. The method of claim 27, wherein a plurality of second primers, each complementary to a segment of a different gene of interest, are used, to generate a plurality of amplification products.

29. The method of claim 18, wherein the polynucleotides are obtained by a method comprising:

(a) reacting cellular chromatin related to the gene of interest with a chemical or enzymatic probe to generate chromatin-associated DNA fragments, wherein the DNA fragments comprise, at their termini, sites of probe reaction which identify accessible regions of cellular chromatin;

(b) attaching a first adapter polynucleotide to the termini generated by the probe to generate adapter-ligated fragments;

(c) digesting the adapter-ligated fragments with a restriction enzyme to generate a population of digested fragments, wherein the population comprises digested fragments having a first end that comprises the first adapter and a second end formed via the activity of the restriction enzyme;

(d) contacting the digested fragments with a primer complementary to the first adapter under conditions wherein the primer is extended to generate a plurality of extension products, each comprising a first end that comprises the first adapter and a second end that can be attached to a second adapter polynucleotide;

(e) joining the second adapter to the second end of each of the plurality of extension products to form a plurality of modified fragments, each of which comprises the first and second adapters at its first and second end, respectively;

(f) amplifying the plurality of modified fragments in the presence of primers complementary to the sequences of the first and second adapters to generate a population of amplified products comprising sequences corresponding to accessible regions of cellular chromatin; and

determining the nucleotide sequences of the amplified products to obtain the polynucleotide sequences.

30. The method of claim 18, wherein:

(a) identifying comprises identifying a functional accessible region and determining whether the functional accessible region comprises a binding site for a transcription factor; and

(b) selecting comprises choosing the functional domain of the exogenous regulatory molecule to be the same as the functional domain of the transcription factor.

31. The method of claim 18, wherein:

(a) identifying comprises identifying a gateway accessible region; and

(b) selecting comprises choosing the DNA binding domain of the exogenous regulatory molecule to specifically bind to a segment of the gateway accessible region.

32. The method of claim 31, wherein the DNA binding domain is a zinc finger DNA-binding domain.

33. The method of claim 18, wherein:

the identifying comprises identifying a gateway accessible region and determining whether the gateway accessible region comprises a binding site for a known transcription factor; and

the selecting comprises choosing the DNA binding domain of the exogenous regulatory molecule to be the same as the DNA binding domain of the known transcription factor.