Potential Transcriptional Potential Transcriptional Activation of the Foxn1 Activation of the Foxn1

Gene by the Runx1 FactorGene by the Runx1 FactorKatelyn SeloffKatelyn Seloff

TAMS, College of Arts and SciencesTAMS, College of Arts and SciencesEli Raveh, Department of Animal and Cell Eli Raveh, Department of Animal and Cell Biology, Hebrew University of JerusalemBiology, Hebrew University of Jerusalem

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BackgroundBackground Question: Could the transcription factor Question: Could the transcription factor Foxn1Foxn1

be a target gene of another transcription factor, be a target gene of another transcription factor, Runx1Runx1??

Reasoning:Reasoning: Overlapping expression patterns (both expressed in Overlapping expression patterns (both expressed in

cuticle and cortex of hair shaft)cuticle and cortex of hair shaft) Similar effectsSimilar effects

• Runx1Runx1 epidermal KO causes impaired hair shaft stability, epidermal KO causes impaired hair shaft stability, hair follicle structure, and differentiation of hair stem cellshair follicle structure, and differentiation of hair stem cells

• Foxn1Foxn1 mutations cause hair fragility and stunted hair mutations cause hair fragility and stunted hair growth; growth; Foxn1Foxn1 seems to be required for assembly of hair seems to be required for assembly of hair shaftshaft

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Commonalities lead to conclusion that the Commonalities lead to conclusion that the genes could be mutually controlled or control genes could be mutually controlled or control each othereach other

Evidence indicates Evidence indicates Foxn1Foxn1 doesn’t regulate doesn’t regulate Runx1Runx1

Foxn1Foxn1 expression assumed not to depend expression assumed not to depend completely on completely on Runx1Runx1 ( (Runx1Runx1 epidermal KO epidermal KO does not result in the severe phenotype seen in does not result in the severe phenotype seen in Foxn1Foxn1 mutations) mutations)

Runx1 partially regulates Foxn1 expression?Runx1 partially regulates Foxn1 expression?

Reasoning (Cont.)Reasoning (Cont.)

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Hypothesis: Hypothesis: Runx1Runx1 positively regulates the positively regulates the expression of expression of Foxn1Foxn1 as as

an activator of thean activator of theproximal promoter of proximal promoter of

Foxn1Foxn1..

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ProceduresProcedures Foxn1Foxn1 fragment amplified by PCR fragment amplified by PCR Amplified Amplified Foxn1Foxn1 fragment and the vector fragment and the vector

plasmid digested by two restriction enzymes, plasmid digested by two restriction enzymes, Kpn1 and Sac1Kpn1 and Sac1 Two controlsTwo controls

• Sac1 digestion not completely efficient (allowed for Sac1 digestion not completely efficient (allowed for background colonies), but both digestions successfulbackground colonies), but both digestions successful

Products quantified (plasmid: 18 ng/μL DNA; Products quantified (plasmid: 18 ng/μL DNA; fragment: 67 ng/μL DNA)fragment: 67 ng/μL DNA) Necessary to plan ligation of amplified Necessary to plan ligation of amplified Foxn1Foxn1

fragment into vector, pGL3-basic (aforementioned fragment into vector, pGL3-basic (aforementioned plasmid)plasmid)

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Procedures (Cont.)Procedures (Cont.) Four ligations carried outFour ligations carried out Ligation products transformed into Ligation products transformed into E. coliE. coli

bacteria by induction of artificial competencebacteria by induction of artificial competence 26 colonies used for mini-cultures, four then 26 colonies used for mini-cultures, four then

used for mini-preparationsused for mini-preparations One colony chosen for a midi-culture, used for One colony chosen for a midi-culture, used for

a midi-preparationa midi-preparation SequencedSequenced

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Plasmid SetupPlasmid SetupSix plasmids in use (1 & 2 Six plasmids in use (1 & 2

pictured at right):pictured at right): 1: pGL3-basic1: pGL3-basic 2: K14-2: K14-Runx1Runx1

Cells express K14 constitutively, Cells express K14 constitutively, so will drive production of so will drive production of Runx1Runx1

3: K14 (control3: K14 (control––no no Runx1Runx1)) 4: pcgn-CBF4: pcgn-CBFΒΒ

Co-binding factor of Co-binding factor of Runx1Runx1, , should increase should increase Runx1Runx1 activity activity

5: pcgn (control5: pcgn (control––no CBFB)no CBFB) 6: Renilla (control for 6: Renilla (control for

transfection efficiency)transfection efficiency)

K14-Runx1 (plasmid 1) and pGL3-basic (plasmid 2) setups

LuciferaseGene encodingRunx1

Runx1 binding site(Foxn1 promoter)

Luciferase transcripts(luminescence)

Runx1

K14 promoter

K14 transcription activators

K14-Runx1 pGL3-basicK14-Runx1

Amp Resistance

Amp Resistance

LuciferaseGene encodingRunx1

Runx1 binding site(Foxn1 promoter)

Luciferase transcripts(luminescence)

Runx1

LuciferaseGene encodingRunx1

Runx1 binding site(Foxn1 promoter)

Luciferase transcripts(luminescence)

Runx1

K14 promoter

K14 transcription activators

K14-Runx1 pGL3-basicK14-Runx1

Amp Resistance

Amp Resistance

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Final SetupFinal SetupTransfection SetupTransfection Setup

WellsWells Contents Contents (K14-Runx1, pcgn, pcgn-CBFB)(K14-Runx1, pcgn, pcgn-CBFB) ControlControl1, 21, 2 NoneNone +K14, -+K14, -Runx1Runx1

3, 43, 4 100ng K14-100ng K14-Runx1Runx1

5, 65, 6 200ng K14-200ng K14-Runx1Runx1

7, 87, 8 300ng K14-300ng K14-Runx1Runx1

9, 109, 10 100ng pcgn-CBFB100ng pcgn-CBFB +pcgn, +CBFB, -+pcgn, +CBFB, -Runx1Runx1

11, 1211, 12 100ng K14-100ng K14-Runx1Runx1, 100ng pcgn-CBFB, 100ng pcgn-CBFB

13, 1413, 14 300ng K14-300ng K14-Runx1Runx1, 100ng pcgn-CBFB, 100ng pcgn-CBFB

15, 1615, 16 300ng K14-300ng K14-Runx1Runx1, 300ng pcgn-CBFB, 300ng pcgn-CBFB

17, 1817, 18 300ng K14-300ng K14-Runx1Runx1, 100ng pcgn, 100ng pcgn +pcgn, -CBFB+pcgn, -CBFB

19, 2019, 20 300ng K14-300ng K14-Runx1Runx1 Repeat of wells 7, 8Repeat of wells 7, 8

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Final Setup (Cont.)Final Setup (Cont.) TransfectionTransfection Luciferase reporter assay runLuciferase reporter assay run

Luc+Luc+ expression measured expression measured RenillaRenilla substrate solution added to stop substrate solution added to stop Luc+Luc+

expression and act as a positive control (expression and act as a positive control (RenillaRenilla needed no ectopic factor for expression)needed no ectopic factor for expression)

Renilla expression measuredRenilla expression measured Ratio between Luc+-dependent luminescence and Ratio between Luc+-dependent luminescence and

Renilla-dependent luminescence calculatedRenilla-dependent luminescence calculated

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Raw data: measurement of luminescence of Raw data: measurement of luminescence of each well from each well from Luc+ Luc+ expression alone, in RLUsexpression alone, in RLUs

No obvious trendsNo obvious trends Did not allow for accurate comparisons Did not allow for accurate comparisons

between wellsbetween wells

Raw DataRaw Data

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Normalized DataNormalized Data

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Normalized Data (Cont.)Normalized Data (Cont.)

Identical

ControlsNo Runx1 vs. 100ng Runx1

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Normalized Data (Cont.)Normalized Data (Cont.)

100ng CBFB no Runx1 vs. 100ng

CBFB + 100ng Runx1

300ng Runx1 no CBFBvs. 300ng Runx1 +

100ng CBFB

100ng Runx1 no CBFB vs. 100ng

CBFB + 100ng Runx1

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ConclusionsConclusions Data collected does not support hypothesis Data collected does not support hypothesis

(expression of Foxn1 is positively regulated by (expression of Foxn1 is positively regulated by Runx1)Runx1)

Based on this experiment, Runx1 does not Based on this experiment, Runx1 does not activate Foxn1 as hypothesizedactivate Foxn1 as hypothesized

These results are inconclusiveThese results are inconclusive This experiment was done in vitroThis experiment was done in vitro KeratinocytesKeratinocytes Only tested 2Only tested 2ndnd transcriptional start site of transcriptional start site of Foxn1Foxn1 Negative regulation of Negative regulation of Foxn1Foxn1 by by Runx1Runx1 still possible still possible

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AcknowledgmentsAcknowledgments Mr. Eli RavehMr. Eli Raveh Dr. Uri GatDr. Uri Gat Legacy Heritage FundLegacy Heritage Fund Hebrew University of JerusalemHebrew University of Jerusalem Aviva SiegelAviva Siegel

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Thank You For Listening!Thank You For Listening!Any Questions?Any Questions?