Dendrite Development Regulated by CREST, a Calcium-Regulated Transcriptional Activator

Science

Volumn 303, Issue 5655, 2004, Pages 197-202

  Aizawa, Hiroyuki ^a   Hu, Shu Ching ^a   Bobb, Kathryn ^a   Balakrishnan, Karthik ^a   Ince, Gulayse ^a   Gurevich, Inga ^a   Cowan, Mitra ^a   Ghosh, Anirvan ^a,b  

^a JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BINDING ENERGY; BRAIN; CALCIUM; CHEMICAL ACTIVATION; GENETIC ENGINEERING; MUTAGENESIS; PROTEINS;

DENDRITIC GROWTH; MORPHOGENESIS;

NEUROLOGY;

CALCIUM RESPONSIVE TRANSACTIVATOR; CYCLIC AMP; CYCLIC AMP RESPONSIVE ELEMENT BINDING PROTEIN BINDING PROTEIN; NUCLEAR PROTEIN; TRANSACTIVATOR PROTEIN; UNCLASSIFIED DRUG;

BRAIN; GENE EXPRESSION;

ANIMAL CELL; ARTICLE; BRAIN CORTEX; BRAIN DEVELOPMENT; CELL ACTIVITY; CELL MATURATION; DENDRITE; GENE DISRUPTION; GENE EXPRESSION; MORPHOGENESIS; MOUSE; NERVE GROWTH; NONHUMAN; PRIORITY JOURNAL; PROTEIN FUNCTION; TRANSCRIPTION INITIATION; TRANSCRIPTION REGULATION;

AMINO ACID SEQUENCE; ANIMALS; BLOTTING, NORTHERN; BRAIN; CALCIUM; CALCIUM CHANNELS; CELL LINE; CELLS, CULTURED; CEREBRAL CORTEX; CLONING, MOLECULAR; CREB-BINDING PROTEIN; DENDRITES; GENE EXPRESSION PROFILING; GENE EXPRESSION REGULATION, DEVELOPMENTAL; GENE LIBRARY; GENE TARGETING; HUMANS; IN SITU HYBRIDIZATION; MICE; MICE, KNOCKOUT; MOLECULAR SEQUENCE DATA; MUTATION; NERVOUS SYSTEM; NEURONS; NUCLEAR PROTEINS; PROTEIN STRUCTURE, TERTIARY; RATS; RECOMBINANT FUSION PROTEINS; TRANS-ACTIVATION (GENETICS); TRANS-ACTIVATORS; TRANSCRIPTION, GENETIC; TRANSFECTION;

ANIMALIA;

EID: 0346969980     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.1089845     Document Type: Article

References (30)

1. L. C. Katz, C. J. Shatz, Science 274, 1133 (1996).
2. A. Ghosh, M. E. Greenberg, Science 268, 239 (1995).
3. E. Nedivi, J. Neurobiol. 41, 135 (1999).
4. A. E. West et al., Proc. Natl. Acad. Sci. U.S.A. 98, 11024 (2001).
5. L. Keegan, G. Gill, M. Ptashne, Science 231, 699 (1986).
6. M. Ptashne, A Genetic Switch (Blackwell Scientific Publications and Cell Press, Cambridge, MA, 1992).
7. To determine the cDNA pool size appropriate for the screen, we used GAL4-CREB as a positive control in sequential dilution experiments. GAL-4-CREB could induce detectable transactivation of UA5-CAT in the presence of competing GAL4-DBD at a ratio of 1:1000, suggesting a pool size of 1000 cDNAs.
8. The library is maintained in the form of 200 pools of 1000 cDNAs, as plasmid DNA as well as bacterial transformants.
9. Cultures were transfected at 3 DIV and stimulated by depolarization with 50 mM KCI overnight at 5 DIV. GAL4-CREB, diluted at 1:1000 with GAL4-DBD served as a positive control.
10. Materials and methods are available as supporting material on Science Online.
11. J. Clark et al., Nature Genet. 7, 502 (1994).
12. N. R. dos Santos et al., Hum. Mol. Genet. 6, 1549 (1997).
13. D. Brett et al., Hum. Mol. Genet. 6, 1559 (1997).
14. C. Thaete et al., Hum. Mol. Genet. 8, 585 (1999).
15. J. E. Eid, A. L. Kung, R. Scully, D. M. Livingston, Cell 102, 839 (2000).
16. R. Kwok et al., Nature 370, 223 (1994).
17. J. R. Lundblad, R. P. S. Kwok, M. E. Laurance, M. L. Harter, R. H. Goodman, Nature 374, 85 (1995).
18. X. Yang, V. V. Ogryzko, J. Nishikawa, B. H. Howard, Y. Nakatani, Nature 382, 319 (1996).
19. A. J. Bannister, T. Kouzarides, Nature 384, 641 (1996).
20. H. Aizawa et al., data not shown.
21. L. Redmond, A. Kashani, A. Ghosh, Neuron 34, 999 (2002).
22. S. Chawla, G. E. Hardingham, D. R. Quinn, H. Bading, Science 281, 1505 (1998).
23. G. E. Hardingham, S. Chawla, F. H. Cruzalegui, H. Bading, Neuron 22, 789 (1999).
24. S.-C. Hu, J. Chrivia, A. Ghosh, Neuron 22, 799 (1999).
25. S.-C. Hu, H. Aizawa, A. Ghosh, unpublished results.
26. MegAlign program, DNASTAR, Madison, WI.
27. L. Redmond, S.-R. Oh, C. Hicks, G. Weinmaster, A. Ghosh, Nature Neurosci. 3, 30 (2000).
28. To create the CREST antibody, We used PCR to amplify full-length crest cDNA from PO mouse brain total RNA and cloned it into pET21 vector for expression of His-tagged CREST protein in bacteria. After isopropyl-β -D-thiogalactopyranoside induction, CREST protein was mainly collected in the inclusion body of the bacteria and extracted with 6 M urea, We further purified the protein with Ni-NTA agarose fractionation. After SD5-polyacrylamide gel electrophoresis (PAGE), the Coomassie brilliant blue-stained CREST band was excised and injected into rabbits to produce anti-CREST antiserum.
29. To generate the crest knockout mice, crest genomic DNA was cloned by screening a 129SVJ mouse genomic phage library (gift of A. Kolodkin) with full-length mouse crest cDNA as a probe. After mapping the restriction enzyme sites, we generated a knockout vector with a Neo gene cassette (Fig, 4). We deleted the poly(A) addition signal from the Neo gene cassette for poly(A) trapping method of gene targeting. The cassette replaced all of exon 4 and a 5′ portion of exon 5. ES cells transfected with the targeting vector and resistant to G418 and gancyclovir were expanded and screened by genomic Southern blotting. Correctly targeted ES cells were injected into C57B6J-derived blastocysts and resulted in the generation of several high-percentage chimeras, which produced germline targeted offspring. Genotyping of mice was performed on tail clip DNA by PCR.
30. We thank D. Livingston for the UAS-CAT construct; A. Lanahan for advice on library construction; A. Kolodkin for rat phage libraries; L. Redmond, A. Datwani, K. Whitford, M.-R. Song, and G. Ince for various procedures; J. Nathans, C. Montell, D. Ginty, P. Worley, S. Snyder, D. Linden, D. Murphy, P. Kim, M. Molliver for discussions; and M. Greenberg, D. Ginty, A. Kolodkin, and S. Snyder for comments on the manuscript. Supported by grants from NIH (MH60598 and NS39993), the March of Dimes Birth Defects Foundation (A.G.), the Klingenstein Foundation (A.G.), and a Merck SchoLar Award (A.G.). H.A. was supported by a Uehara Memorial Foundation Research Fellowship.