1. Potential Transcriptional Activation of the Foxn1 Gene by the Runx1 Factor Katelyn Seloff TAMS, College of Arts and Sciences Eli Raveh, Department of Animal and Cell Biology, Hebrew University of Jerusalem

2. 2Background  Question: Could the transcription factor Foxn1 be a target gene of another transcription factor, Runx1?  Reasoning: Overlapping expression patterns (both expressed in cuticle and cortex of hair shaft) Overlapping expression patterns (both expressed in cuticle and cortex of hair shaft) Similar effects Similar effects Runx1 epidermal KO causes impaired hair shaft stability, hair follicle structure, and differentiation of hair stem cellsRunx1 epidermal KO causes impaired hair shaft stability, hair follicle structure, and differentiation of hair stem cells Foxn1 mutations cause hair fragility and stunted hair growth; Foxn1 seems to be required for assembly of hair shaftFoxn1 mutations cause hair fragility and stunted hair growth; Foxn1 seems to be required for assembly of hair shaft

3. 3  Commonalities lead to conclusion that the genes could be mutually controlled or control each other  Evidence indicates Foxn1 doesn’t regulate Runx1  Foxn1 expression assumed not to depend completely on Runx1 (Runx1 epidermal KO does not result in the severe phenotype seen in Foxn1 mutations)  Runx1 partially regulates Foxn1 expression? Reasoning (Cont.)

4. 4 Hypothesis: Runx1 positively regulates the expression of Foxn1 as an activator of the proximal promoter of Foxn1.

5. 5 Procedures  Foxn1 fragment amplified by PCR  Amplified Foxn1 fragment and the vector plasmid digested by two restriction enzymes, Kpn1 and Sac1 Two controls Two controls Sac1 digestion not completely efficient (allowed for background colonies), but both digestions successfulSac1 digestion not completely efficient (allowed for background colonies), but both digestions successful  Products quantified (plasmid: 18 ng/μL DNA; fragment: 67 ng/μL DNA) Necessary to plan ligation of amplified Foxn1 fragment into vector, pGL3-basic (aforementioned plasmid) Necessary to plan ligation of amplified Foxn1 fragment into vector, pGL3-basic (aforementioned plasmid)

6. 6 Procedures (Cont.)  Four ligations carried out  Ligation products transformed into E. coli bacteria by induction of artificial competence  26 colonies used for mini-cultures, four then used for mini-preparations  One colony chosen for a midi-culture, used for a midi-preparation Sequenced Sequenced

7. 7 Plasmid Setup Six plasmids in use (1 & 2 pictured at right):  1: pGL3-basic  2: K14-Runx1 Cells express K14 constitutively, so will drive production of Runx1 Cells express K14 constitutively, so will drive production of Runx1  3: K14 (control–no Runx1)  4: pcgn-CBFΒ Co-binding factor of Runx1, should increase Runx1 activity Co-binding factor of Runx1, should increase Runx1 activity  5: pcgn (control–no CBFB)  6: Renilla (control for transfection efficiency) K14-Runx1 (plasmid 1) and pGL3-basic (plasmid 2) setups

8. 8 Final Setup Transfection Setup Wells Contents (K14-Runx1, pcgn, pcgn-CBFB) Control 1, 2 None +K14, -Runx1 3, 4 100ng K14-Runx1 5, 6 200ng K14-Runx1 7, 8 300ng K14-Runx1 9, 10 100ng pcgn-CBFB +pcgn, +CBFB, -Runx1 11, 12 100ng K14-Runx1, 100ng pcgn-CBFB 13, 14 300ng K14-Runx1, 100ng pcgn-CBFB 15, 16 300ng K14-Runx1, 300ng pcgn-CBFB 17, 18 300ng K14-Runx1, 100ng pcgn +pcgn, -CBFB 19, 20 300ng K14-Runx1 Repeat of wells 7, 8

9. 9 Final Setup (Cont.)  Transfection  Luciferase reporter assay run Luc+ expression measured Luc+ expression measured Renilla substrate solution added to stop Luc+ expression and act as a positive control (Renilla needed no ectopic factor for expression) Renilla substrate solution added to stop Luc+ expression and act as a positive control (Renilla needed no ectopic factor for expression) Renilla expression measured Renilla expression measured Ratio between Luc+-dependent luminescence and Renilla-dependent luminescence calculated Ratio between Luc+-dependent luminescence and Renilla-dependent luminescence calculated

10. 10  Raw data: measurement of luminescence of each well from Luc+ expression alone, in RLUs  No obvious trends  Did not allow for accurate comparisons between wells Raw Data

11. 11 Normalized Data

12. 12 Normalized Data (Cont.) Identical Controls No Runx1 vs. 100ng Runx1

13. 13 Normalized Data (Cont.) 100ng CBFB no Runx1 vs. 100ng CBFB + 100ng Runx1 300ng Runx1 no CBFB vs. 300ng Runx1 + 100ng CBFB 100ng Runx1 no CBFB vs. 100ng CBFB + 100ng Runx1

14. 14Conclusions  Data collected does not support hypothesis (expression of Foxn1 is positively regulated by Runx1)  Based on this experiment, Runx1 does not activate Foxn1 as hypothesized  These results are inconclusive This experiment was done in vitro This experiment was done in vitro Keratinocytes Keratinocytes Only tested 2 nd transcriptional start site of Foxn1 Only tested 2 nd transcriptional start site of Foxn1 Negative regulation of Foxn1 by Runx1 still possible Negative regulation of Foxn1 by Runx1 still possible

15. 15Acknowledgments  Mr. Eli Raveh  Dr. Uri Gat  Legacy Heritage Fund  Hebrew University of Jerusalem  Aviva Siegel

16. 16 Thank You For Listening! Any Questions?