Artificial transcription factors and uses thereof
The present invention relates to artificial transcription factors (ATFs) that alter gene expression, including inducing pluripotency in cells or promoting the conversion of cells to specific cell fates. In particular, provided herein is a zinc-finger based ATF library that can be screened in cells by looking for expression of a specific gene (e.g., reporter expression), monitoring for cell surface markers or morphology, or via functional assays.
1. An artificial transcription factor comprising a polydactyl zinc finger protein comprising an interaction domain, an activation domain, and two zinc finger domains, each of the two zinc finger domains comprising a variable domain independently encoded by nucleic acid sequence of SEQ ID NO:2 (VNN-TCC-VNN-VNN-CTC-ACC-VNN), wherein each VNN of SEQ ID NO:2 is a codon corresponding to an amino acid selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V, with the proviso that the variable domain in at least one of the two zinc finger domains has a sequence of one of SEQ ID NOS:123-341.
2. The artificial transcription factor of claim 1 , wherein the polydactyl zinc finger protein comprises three zinc finger domains encoded by SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, wherein each VNN of SEQ ID NOS:4, 5, and 6 is a codon corresponding to an amino acid selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V.
3. The artificial transcription factor of claim 1 , wherein the interaction domain comprises a 15-amino acid peptide that enables interaction with the hydrophobic face of the most N-terminal zinc finger domain.
4. The artificial transcription factor of claim 1 , wherein the activation domain comprises four tandem repeats of DALDDFDLDML (SEQ ID NO:7).
5. The artificial transcription factor of claim 1 , further comprising a nuclear localization signal.
6. The artificial transcription factor of claim 5 , wherein the nuclear localization signal is encoded by an amino acid sequence selected from the group consisting of KDKKADKSVV (SEQ ID NO:11) and PKKKRKV (SEQ ID NO:12).
7. A method of reprogramming a somatic cell to pluripotency, wherein the method comprises:
(a) exposing a somatic cell to a plurality of artificial transcription factors (ATFs), wherein the plurality of ATFs comprises at least one of ZFATF1 and ZFATF2;
(b) further exposing the somatic cell to a plurality of potency determining factors comprising Sox2, Klf4, and c-Myc; and
(c) culturing the exposed cells to obtain reprogrammed cells having a higher potency level than the somatic cell.
8. The method of claim 7 , wherein the plurality of ATFs comprises ZFATF1, ZFATF2, and ZFATF5.
9. The method of claim 7 , wherein the plurality of ATFs comprises ZFATF1 and ZFATF2.
10. A method of directing differentiation of a pluripotent stem cell to a somatic cell, wherein the method comprises;
(a) exposing a pluripotent stem cell to one or more artificial transcription factors (ATFs), wherein, to produce a cardiomyocyte somatic cell, the one or more ATFs comprise at least one of ZFATF1 and ZFATF2, and wherein, to produce a hematopoietic somatic cell, the one or more ATFs comprise at least one of ZFATF20, ZFATF21, ZFATF1, ZFATF6, ZFATF10, ZFATF13, ZFATF17, ZFATF22, ZFATF23, ZFATF24, ZFATF25, ZFATF26, ZFATF27, ZFATF28, ZFATF29, and ZFATF30; and
(b) culturing the exposed cells of (a) for about 7-10 days, wherein to produce a cardiomyocyte somatic cell, the exposed cells are cultured in the presence of a Wnt activator, and wherein, to produce a hematopoietic somatic cell, the exposed cells are cultured in a basal culture medium comprising FGF2, SCF, and thrombopoietin, and in the presence of TAL1.
11. The method of claim 10 , wherein the ATFs comprise ZFATF1, ZFATF2, and ATF5.
12. The method of claim 10 , wherein the ATFs comprise ZFATF19, ZFATF20, ZFATF21, and ATF5, and wherein a cell population comprising erythroid-like cells is produced.
13. The method of claim 10 , wherein the ATFs comprise ZFATF1, ZFATF6, ZFATF10, ZFATF13, ZFATF17, ZFATF22, ZFATF23, ZFATF24, ZFATF25, ZFATF26, ZFATF27, ZFATF28, ZFATF29, ZFATF30, and ZFATF31, and wherein a cell population comprising monocyte-like cells is produced.
14. A method for preparing a zinc finger-based artificial transcription factor (ATF) library comprising generating a plurality of elements, whereby each element of said ATF library comprises:
a zinc finger backbone comprising two oligonucleotides, each of the two oligonucleotides encoding SEQ ID NO:3 (X1-S-X2-X3-L-T-X6);
an interaction domain-encoding sequence; and
an activation domain-encoding sequence,
wherein X1 is an amino acid selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V; X2 is selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V; X3 is selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V; and X6 is selected from the group consisting of R, H, K, D, Q, S, T, N, E, G, P, A, I, L, M, and V, wherein at least one element encodes the artificial transcription factor of claim 1 .
15. The method of claim 14 , wherein the ATF library has a complexity of about 2.62×10 6 .
16. The method of claim 14 , wherein the interaction domain comprises a 15-amino acid peptide that enables interaction with the hydrophobic face of the most N-terminal zinc finger domain.
17. The method of claim 14 , wherein the activation domain comprises four tandem repeats of DALDDFDLDML (SEQ ID NO:7).
18. A zinc finger-based artificial transcription factor (ATF) library obtained according to the method of claim 14 .
19. The artificial transcription factor of claim 1 , wherein the variable domain in each of the two zinc finger domains has a sequence of one of SEQ ID NOS:123-341.
20. The artificial transcription factor of claim 1 , wherein the variable domain in at least one of the two zinc finger domains has a sequence of one of SEQ ID NOS:123-147.
21. The artificial transcription factor of claim 1 , wherein the variable domain in each of the two zinc finger domains has a sequence of one of SEQ ID NOS:123-147.
22. The artificial transcription factor of claim 1 , wherein the variable domain in at least one of the two zinc finger domains has a sequence of one of SEQ ID NOS:123-128, 131, 136, and 138-145.
23. The artificial transcription factor of claim 1 , wherein the variable domain in each of the two zinc finger domains has a sequence of one of SEQ ID NOS:123-128, 131, 136, and 138-145.
24. The artificial transcription factor of claim 1 , wherein the variable domain in at least one of the two zinc finger domains has a sequence of one of SEQ ID NOS: 123, 124, 131, 136, 139, 142, and 144.