Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.com 20 cusjVolume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu N EU RO SC IE N C E cusjcolumbia undergraduate science journalResearch Articles 21cusj Volume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu M O LEC U LA R BIO LO G Y Identifying serum-inducible sequence elements on the mouse ZFP36 promoter Max Shutran1,*, Ron Prywes 2,* 1Tufts University School of Medicine, Boston, MA 02110, 2Department of Biological Sciences, Columbia University, New York, NY 10027 APUJ�ÄUNLY�WYV[LPU�����A-7����PZ�HU�95(�IPUKPUN�WYV[LPU�[OH[�KV^UYLN\SH[LZ�[OL�[YHUZSH[PVU�VM�JLY[HPU�NLUL�WYVK\J[Z�I`� inducing the degradation of mRNA. ZFP36 is an immediate-early gene: its transcription is activated directly following ap- propriate cellular stimuli, without any need for intervening protein synthesis. Prominent mRNA targets of ZFP36 are tumor ULJYVZPZ�MHJ[VY����;5-�����LWPKLYTHS�NYV^[O�MHJ[VY��,.-��HUK�J�MVZ��(Z�KLMLJ[Z�PU�[OL�J�MVZ�HUK�,.-�WH[O^H`Z�OH]L�ILLU� consistently found in cancers, ZFP36 is thought to be a tumor-suppressor. ZFP36 also is responsible for the suppression VM�;5-����NP]PUN�P[�HU�HKKP[PVUHS�YVSL�PU�[OL�JVU[YVS�VM�[OL�PUÅHTTH[VY`�PTT\UL�YLZWVUZL��;O\Z��Z[\K`PUN�[OL�YLN\SH[PVU� HUK�HJ[PVU�VM�A-7���TH`�`PLSK�PUZPNO[Z�PU[V�TLJOHUPZTZ�\UKLYS`PUN�JHUJLY�HUK�PUÅHTTH[VY`�Z`UKYVTLZ��;V�L_HTPUL�[OL� regulation of ZFP36, two conserved regions upstream of the start of transcription were assayed for their ability to confer serum induction. Using a luciferase reporter gene system in HeLa cells, we show that neither region examined is induced by serum. Key words: electron transfer, photosynthesis, quinones, UV-Vis spectroscopy Immediate-early genes are characterized by immediate transcriptional activation upon appropriate stimulation, including treatment of cells with mitogen-containing se- rum, without any need for intervening protein synthesis (Johansen, 1995). As they are among the !rst genes to be activated in many signaling pathways, immediate-early genes provide a gateway to study the cellular response. Zinc !nger protein 36 (ZFP36) is an immediate-early gene product that regulates the expression of some genes by destabilizing their mRNA. "e protein binds to AU-rich sequences on mRNA and catalyzes removal of the poly- adenylated tail, reducing the half-life of the RNA (Blacks- hear, 2002). "e zinc !nger motif is common in DNA and RNA binding proteins, though ZFP36 is unusual in that it has a Cys3-His zinc-coordinating domain, as opposed to the more common Cys2-His2 domain (Tchen, 2004). "is slight di#erence in peptides might subtly impact the nu- cleotide binding dynamics of ZFP36. Notable genes regu- lated in part by ZFP36 include tumor necrosis factor-Ơ� (TNF-Ơ), epidermal growth factor (EGF) and c-fos, a tran- scription factor that promotes proliferation (Amit, 2007) Mice de!cient in ZFP36 develop an excess of TNF-Ơ, and the overabundance of this cytokine results in autoimmune complications such as arthritis (Lai, 1998). As EGF and c-fos are both implicated in proliferation, ZFP36 is also thought to act as a tumor-suppressor (Amit, 2007) "e ZFP36 gene is regulated in several ways. Tchen and colleagues et al. have shown that the ZFP36 gene is mito- gen-inducible via protein kinase p38 (Tchen, 2004). "is result is consistent with qPCR data demonstrating that ZFP36 is induced (upregulated) strongly by serum after one hour (Figure 1). However, the upregu- lation seems to be independent of serum response factor (SRF), the transcription factor that primarily mediates the serum response (Prywes, unpublished data). Lai and colleages showed that there are im- portant regulatory elements downstream of the start of ZFP36 transcription, and at least one of those regulatory elements is on an intron (Lai, 1995; Lai, 1998). Given that ZFP36 is induced by serum one hour after induction, we hypothesized that there may be additional serum-dependent regulatory elements upstream of the start of transcription. We identi!ed two conserved regions in the promoter region of mouse ZFP36 and used a luciferase reporter gene assay to test the ability of each region to confer serum induction in HeLa cells. However, we ultimately ascertained that neither region was able to confer serum induction of ZFP36 in HeLa cells. "is study and other studies of the regulation and action of ZFP36 will provide vital information about this protein, and further research will play a signi!cant role in gaining a better understanding of the biological pathways for prolif- eration and in$ammation. Introduction Copyright: © 2009 The Trustees of Columbia University, Co- lumbia University Libraries, some rights reserved, Albert, et al. Received Dec. 29, 2009. Accepted Feb. 8, 2009. Published April 5, 2009. *To whom correspondence should be addressed: Columbia University, Department of Biology. 813B Fairchild Center. Neew York, NY 10027 Max.Shutran@tufts.edu mrp6@columbia.edu the connections to appear unclear. By stereotaxically injecting virus we can also be more accurate and consis- tent with future injections into the mouse brain. Dif- ferent orientations of slicing must be tested in order to find the optimal orientation for preserving connections. Once the connection from CA1 to the EC is clearly es- tablished, we also plan to examine the connections pro- jecting from the EC to CA1. This anatomical data will contribute to the success of future electrophysiological studies. I would like to thank Dr. Siegelbaum for giving me the amazing opportunity to perform research in his lab and Rebecca Piskorowski for her helpful advice, con- stant guidance, and patience. I would also like to thank Justine Barry for her frequent and important support, guidance, and instruction, Sebastian Thuault for one of his mouse brains, and the rest of the Siegelbaum Lab. Buzsaki, G. 1989. Two-stage model of memory trace formation: A role for “noisy” brain states. Neu- rosci 31: 551-570. de Curtis, M., D. Paré, and R.R. Llinás. The elec- trophysiology of the olfactory-hippocampal circuit in the isolated and perfused adult mammalian brain in vitro. Hippocampus. 1991 Oct;1(4):341-54. Eichenbaum, H., A.P. Yonelinas, and C. Ranga- nath. “The Medial Temporal Lobe and Recognition Mem- ory.” Annu. Rev. Neurosci. 2007.30:123-152. Eldridge, L.L., B.J. Knowlton, C.S. Furmanski, S.Y. Bookheimer, S.A. Engel. Nat Neurosci. Remembering epi- sodes: a selective role for the hippocampus during retriev- al. 2000 Nov;3(11):1149-52. Franklin, Keith B. J., and George Paxinos. The Mouse Brain in Stereotaxic Coordinates, Third Edition. New York: Academic, 2007. Godement, P., J. Vaselow, S. Thanos, and F. Bonhoeffer. (1987) A study in developing visual systems with a new method of staining neurons and the processes in fixed tis- sue. Development, 101: 697-713. Heredia, M., M. Santacana, and F. Valverde. A method of using DiI to study the connectivity of cortical trans- plants. Journal of Neurosci Methods. 36 (1991) 17-25. Honig, M.G. and R.I. Hume (1985) Interactions be- tween sympathetic preganglionic neurons and sympathetic ganglion neurons in vitro. Neurosci. Abstr. 11:98. Hyman, B.T., G.W. Van Hoesen, A.R. Damasio, and C.L. Barnes. Alzheimer’s disease: Cell-specific pathology isolates the hippocampal formation. Science 225: 1168- 1171. Johnston, Daniel, and David G. Amaral. “Hippocam- pus.” The Synaptic Organization of the Brain. Ed. Gordon M. Sheperd. 4th ed. New York: Oxford UP, 1998. Print. Naber, P. A., F. H. Lopes da Silva, M. P. Witter. Re- ciprocal Connections Between the Entorhinal Cortex and Hippocampal Fields CA1 and the Subiculum Are in Reg- ister With the Projections From CA1 to the Subiculum. Hippocampus. 11 (2001) 99-104. O’Keefe, John. A review of the hippocampal place cells. Prog Neurobiol 13: 419–439, 1979. Tamamaki, N. and Y. Nojyo. (2003) Preservation of to- pography in the connections between the subiculum, field CA1, and the entorhinal cortex in rats. Journal of Com- parative Neurology, 353: 379-390. van Groen, T., I. Kadish, and J. M. Wyss. Species dif- ferences in the projections from the entorhinal cortex to the hippocampus. Brain Res Bull. 2002 Feb-Mar 1;57(3- 4):553-6. van Groen, T., P. Miettinen, and I. Kadish. The en- torhinal cortex of the mouse: organization of the pro- jection to the hippocampal formation. Hippocampus. 2003;13(1):133-49. Vindal-Sanz, M., M.P. Villegas-Pérez, G.M. Bray, and A.J. Aguayo. (1988) Persistent retrograde labeling of adult rat retinal ganglion cells with carbocyanine dye DiI. Exp. Neurol., 102:92-101. Witter, M.P., Floris G. Wouterlood, Pieterke A. Naber, and Theo Van Haeften. Anatomical organization of the parahippocampal-hippocampal network. Ann N Y Acad Sci. 2000 Jun;911:1-24. Zola-Morgan S., L.R. Squire, and D. G. Amaral. Hu- man amnesia and the medial temporal region: enduring memory impairment following a bilateral lesion limit- ed to field CA1 of the hippocampus. J Neurosci. 1986 Oct;6(10):2950-67. References (JRUV^SLKNLTLU[Z Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.com 22 cusjVolume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu M O LE C U LA R BI O LO G Y cusjcolumbia undergraduate science journalResearch Articles 23cusj Volume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu M O LEC U LA R BIO LO G Ypriate pH, DNA binds weakly to the calcium phosphate particles, and cells which endocytose the precipitate may take up the genetic material. Each transfection comprised a reporter plasmid, an RL- SV40 plasmid, and the non-luciferase plasmid pCAN, thus keeping the total DNA quantity constant at approxi- mately 1 µg/µL. !e RL-SV40 plasmid carries the enzyme renilla luciferase, which is coupled to a promoter from the SV40 virus. Since renilla luciferase can be measured inde- pendently of "re#y luciferase, and the SV40 promoter is expressed constitutively, this plasmid was used to control for variable transfection e$ciencies. In one experiment, the alternate renilla plasmid RL-TK was used, which has a tyrosine kinase promoter in place of the SV40 promoter. Cells were plated onto 24-well plates the evening prior to transfection at a density of 6.5 x 104 cells per well, in Dulbecco’s Modi"ed Eagle Medium (DMEM) contain- ing 10% NCS. Transfection mixtures were prepared using 1.75 µL of 2.5 M calcium chloride, the appropriate plas- mids, and water to a total volume of 16 µL. !e reporter plasmids were added at a concentration of 125 ng per well, and the renilla plasmid at a concentration of 66 ng per well. For each reporter gene, four identical wells were prepared to allow for duplicates. Each transfection mixture had a total volume of 64 µL. To each tube, 64 µL of HBS (a pH-adjusted mixture containing soluble phosphate salts) was added. !e tubes were vor- texed brie#y and allowed to sit for 10 minutes at room temperature to allow the calcium phosphate to precipitate. !en 32 µL of each mixture was added to the appropriate well. !e following morning, the media was replaced with 10% NCS in DMEM. Serum induction and luciferase assay On the afternoon of the day following transfection, the media in all of the wells were changed to 0.2% neonatal calf serum (NCS). !e following morning, the cells in half of the wells were induced by adding 125 µL of pure NCS, raising the serum concentration to 20%. !ree hours after serum induction, all cells were washed with sterile PBS and treated with 100 µL of Passive Lysis Bu%er, which keeps cytosolic proteins intact during lysis. !e cells were left at room temperature for 20 minutes on a slowly rotating shaker. A bioluminescence detector was used to assay luciferase activity. Two reagents were used for the assay: Luciferase Materials and Methods Plasmid construction Two plasmids, pZFP-2791-FGL3 and pZFP-1835- FGL3, were constructed from a plasmid called pZFP-3.7- FGL3 that had been made previously. !e insert was ex- cised with two di%erent enzyme combinations: KpnI and HindIII in one case, and KpnI and BglII in the other. !is resulted in linearized plasmids that carried an ampicillin resistance gene, a minimal promoter from the human c- fos gene, and a "re#y luciferase gene. !e two promoter regions of interest were obtained from PCR ampli"cation of mouse genomic DNA. !e primers delineated regions from -2591 to -1810 and from -1835 to -901 base pairs relative to the start of transcription, with regions chosen based on mammalian conservation patterns (Figure 3). Each primer was designed with overhangs to insert the re- striction sequences into the PCR products. Each pair of plasmid and PCR product was ligated at 14 ˚C for four hours using the T4 DNA ligase enzyme. !e resultant plasmids were transformed into E. coli for ampli"cation. Sequencing revealed that the pZFP- 1835-FGL3 plasmid had only a single-base change in the c-fos minimal promoter region. !e pZFP-2590-FGL3 se- quence showed an extra, non-contiguous piece of mouse ZFP36 DNA that had been inserted due to the presence of an internal BglII site in the mouse gene. !is piece was excised and the plasmid ampli"ed to give the correct se- quence. !e other plasmids transfected were pCyr61, a control for which moderate serum induction was expected (Babic et al.), pOFGL3, which contains only the c-fos minimal promoter and luciferase, and two other plasmids (pZFP- 3.7-FGL3 and pZFP-932-FGL3), which incorporate re- gions of the mouse promoter at -3700 and -932 bases re- spectively. Transfection Transfection was carried out using a calcium phosphate protocol. In this method, a "ne calcium phosphate precip- itate introduces genetic material into cells. At the appro- � Figure 1 9LZ\S[Z�MYVT�X7*9�MVY�J�MVZ��TPN���HUK�A-7����PU�/L3H�JLSSZ� � Figure 2 .YHWO�PSS\Z[YH[PUN�[OL�PUK\J[PVU�VM�LHJO�YLWVY[LY�NLUL�I`�ZLY\T��;OL�YH^�KH[H�JVUZPZ[LK�VM�K\WSPJH[L�WHPYZ�VM������HUK�����ZLY\T�JVUKP- [PVUZ��NP]LU�HZ�YH[PVZ�VM�3(9�[V�:.�S\TPULZJLUJL��;OL�]HS\LZ�^LYL�JHSJ\SH[LK�I`�UVYTHSPaPUN�LHJO�KH[H�ZL[�[V�VUL�VM�[OL������YH[PVZ��[OLU� H]LYHNPUN�[VNL[OLY�[OL�K\WSPJH[LZ�HUK��PM�H]HPSHISL��V[OLY�KH[H�ZL[Z�MYVT�KPMMLYLU[�KH[LZ��5V�ZPNUPÄJHU[�ZLY\T�PUK\J[PVU�PZ�ZLLU��L_JLW[�PU�[OL� A-7������JVUKP[PVU��^OPJO�\ZLK�93�;2�PU�WSHJL�VM�93�:=���HZ�[OL�YLUPSSH�WSHZTPK� Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.com 24 cusjVolume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu M O LE C U LA R B IO LO G Y cusjcolumbia undergraduate science journalResearch Articles 25cusj Volume 5Spring 2011 Columbia Undergraduate Sci J http://cusj.columbia.edu M O LEC U LA R B IO LO G Y Activating Reagent (LAR) and Stop & Glow reagent (SG). LAR is the substrate of the !re"y luciferase enzyme, and SG is the substrate of renilla luciferase. Each well was measured in two steps. First, 5 µL of cell lysate is added to 25 µL of LAR, and the luminescence was measured. Second, 25 µL of SG was added to the mixture and the luminescence is measured again. When analyzing the data, each measurement was normalized by computing the ratio of LAR to SG luminescence. ZFP36 mRNA was signi!cantly induced by serum Quantitative PCR was run on cDNA prepared from HeLa cells that were stimulated with serum for durations between 0 and 120 minutes (Figure 1). ZFP36 showed an induction pattern similar to c-fos, with strongest induc- tion between 30 minutes and 1 hour. Mig6, a gene that is not induced by serum, was used as a negative control. None of the reporter genes tested was signi!cantly in- duced by serum Figure 2 shows a summary of the results of the lucifer- ase assays performed on transfected HeLa cells. None of the reporter genes showed an appreciable increase in ex- pression upon induction with 20% serum. Minimal serum induction was observed when a di#erent renilla plasmid was used (see Discussion). $e data do not support the hypothesis that conserved regions between -2150 and -901 confer serum induction of ZFP36. $is result was reproducible through several rounds of transfections. $is suggests that the observed se- rum induction of ZFP36 is mediated entirely by elements that are downstream of the start of transcription. One issue that arose in the luciferase data was the in- consistency of renilla measurements. $e SG values tended to vary, and previous experiments using a similar protocol indicated that renilla itself may be induced by serum. To test if serum induction of the renilla gene was a#ecting the results, an alternate renilla gene with a tyrosine kinase promoter (RL-TK) was substituted for RL-SV40 in one trial. $e calculated data do indeed show that pZFP-3.7- FGL3 was induced slightly less than threefold in this trial, but both the LAR and SG measurements from this trial were so di#erent from those in other trials that no conclu- sions could be drawn from this result. $e great variation in LAR measurements indicates a non-trivial relationship between two transfected genes: that is, the presence of one gene in the transfection mixture can a#ect the e%ciency of the transfection of other genes. Another possible con- founder could have been the plasmid preparations them- selves, as calcium phosphate transfection is pH-sensitive. Using a di#erent transfection method could more conclu- sively resolve this issue. Nevertheless, the signi!cant serum induction observed in the qPCR results does not appear to be mediated by any of the upstream regions that were examined. In conclusion, neither region tested was induced by se- rum when expressed on a reporter gene in HeLa cells. Fu- ture studies may further investigate the downstream regu- latory elements characterized by Lai et al., or attempt to !nd other cellular stimuli that regulate ZFP36. Amit, Ido, Ami Citri, Tal Shay et al. “A module of nega- tive feedback regulators de!nes growth factor signaling.” Nature Genetics. 2007, 39(4): 503-512. Babic, Alexander M, Maria L Kireeva, Tatiana V Kole- snikova & Lester F Lau. “CYR61, a product of a growth factor-inducible immediate early gene, promotes angio- genesis and tumor growth.” Proceedings of the National Academy of Sciences United States of America. 1998, 95(11): 6355-6360. Blackshear, Perry J. “Tristetraprolin and other CCCH tandem zinc-!nger proteins in the regulation of mRNA turnover.” Biochemical Society. 2002, 30(6): 945-952. Cen, Bo, Ahayla Selvaraj & Ron Prywes. “Myocardin/ MKL Family of SRF Coactivators: Key Regulators of Im- mediate Early and Muscle Speci!c Gene Expression.” Jour- nal of Cellular Biochemistry. 2004, 93:74-82. Johansen Finn-Eirik & Ron Prywes. “Serum response factor: Transcriptional regulation of genes induced by growth factors and di#erentiation.” Biochimica et Bio- physica Acta. 1995, 1242:1-10. Lai, Wi S, Michael J $ompson & Perry J Blackshear. “Characteristics of the Intron Involvement in the Mitogen- induced Expression of Zfp-36.” $e Journal of Biological Chemistry. 1998, 273: 506-517. Lai, Wi S, Michael J $ompson, Gregory A Taylor, Yi Lui & Perry J Blackshear. “Promoter Analysis of Zfp-36, the Mitogen-inducible Gene Encoding the Zinc Finger Protein Tristetraprolin.” $e Journal of Biological Chemis- try. 1995, 270: 25266-25272. Tchen Carmen R, Matthew Brook, Jeremy Saklatvala & Andrew R Clark. “$e Stability of Tristetraprolin mRNA is Regulated by Mitogen-activated Protein Kinase p38 and by Tristetraprolin Itself.” $e Journal of Biological Chem- istry. 2004, 279: 32393-32400. Discussion and Conclusion Results � Figure 3 Upstream ZFP36 promoter region, showing mammalian conservation regions. From USCS Genome Biogenetics, http://genome.ucsc.edu/ References