Thesis Defense day before

IDENTIFICATION OF INTERACTIONS BETWEEN LHX2

AND OTHER NEOCORTICAL PROTEINS

Will JacksonMaster’s Defense Talk

March 17, 2015

Graded Expression: A gene being turned on in a high to low gradient.

Neocortical Arealization: The process of dividing the neocortex into functional units.

Transcription Factors: Class of proteins that regulate the turning on and off of specific genes.

Basic topics

Neocortex

• Mammalian• Positions of areas are

conserved between species

• This occurs during formation of neocortex (corticogenesis).

Yellow = auditory area

Red = motor area

Green = somatosensory area

Violet = visual area

adapted from Sahara, et al. (2007) Neuron 56:252.high posterior-medial (P-M) to low anterior-lateral (A-L)

Transcription Factors

Turn genes on and off

Work with other proteins to regulate transcription

Regulates arealization

Lhx9, from the Protein Data Bank.

Lhx2

406 amino acids, 44 kDa

A Lim homeodomain protein.

Involved in development of eye, olfactory cortex, spinal cord, hippocampus, and neocortex.

From: The Atlas of Mouse Development

Mice are born 19 days after conception.

7-day old (P7) mouse

Constitutive knock-out Lhx2 mice die at E15.5.

Specific areas aren’t discernable until 7 days after birth.

Lhx2

V1 V2

Transcription factors Lhx2, Sp8, Pax6, Coup-TFI and Emx2 are expressed in gradients.

These gradients determine daughter cell fate.

Daughter cells migrate from ventricular zone (VZ).

Cells migrate into the cortical plate (CP) until they reach the marginal zone (MZ).

Neocortical development

Radial glia

Summary of Cortical Neurogenesis

Summary of Cortical Neurogenesis

Summary of Cortical Neurogenesis

Summary of Cortical Neurogenesis

Summary of Cortical Neurogenesis

Summary of Cortical Neurogenesis

• When Lhx2 is not expressed in the neocortex:

• The neocortex fails to grow to the correct size.

• The piriform cortex grows to compensate for the loss of neocortex.

Lhx2 specifies regional fate in Emx1 lineage of telencephalic progenitors generating cerebral cortex Nat Neurosci. 2009 Nov; 12(11): 1381–1389. Published online 2009 Oct 11.

Lhx2 Mutants

Methods and Results

The Gal4 protein is a transcription factor that activates transcription of yeast reporter genes.

Gal4 contains two domains, each with independent function.

By fusing a bait protein with the Gal4 BD and interacting it with a Prey-Gal4 AD…. we can identify protein-protein interactions.

Yeast Two-Hybrid

• pGBKT7 selects for SD/-Trp• pGADT7 selects for SD/-Leu

• Contains both plasmids selects for SD/-2(-Trp,-Leu)

Selects for protein-protein interactions

SD/-His

SD/-Ade

SD/A

SD/X (blue and white screening

Plasmids Used

Lane 1: pGBKT7-Lhx2 and pGADT7-Lbdv1 Lane 2: pGADT7-Lbdv1Lane 3: pGADT7-T and pGBKT7-53. Lane 4: Ladder

• Lane 1 shows pGBKT7-Lhx2 @ 64 kDa• Lane 3 shows pGBKT7-53 @ 51 kDa• Confirms Lhx2 produced as full length protein.

tRNA Percent used in Mouse

Percent used in Yeast

TTA (L) 6.5% 26.4%

TTG (L) 13.3% 27.1%

CTT (L) 13.2% 12.2%

CTC (L) 20.3% 5.4%

CTA (L) 8.0% 13.4%

CTG (L) 40.0% 10.4%

Confirmation of Lhx2 Production

0 1 2 3 4 5 6 7 80.1

1

10

f(x) = 0.0889969411175737 exp( 0.340394360794436 x )R² = 0.966514042346216

f(x) = 0.239053218069029 exp( 0.261898349079607 x )R² = 0.979428949887578

Control Exponential (Control)Lhx2 Logarithmic (Lhx2)Exponential (Lhx2)

Hours

Log2

(OD

600n

m)

• Doubling time of control (pGBKT7-53) = 2.64 hours • Doubling time of pGBKT7-Lhx2 = 2.32 hours

Lhx2 is not toxic to yeast cells

• Lhx2 and CLIM2 are known to interact.

• Yeast Two-Hybrid with:• GADT7-Lhx2• GBKT7-CLIM2

• Confirms that Lhx2 is folded properly.

Lhx2 Folds Properly

Prey Library yeast

Bait (Lhx2) yeast

-Leucine -Tryptophan

-2 plates (-leu and –trp)-4+A+X plates (-leu, –trp, -ade, -his, aureobasidin A, and X-gal)

Yeast Two-Hybrid Screen

• Streaked yeast onto SD/-leu plates.• Extracted library plasmids from yeast.• Transformed plasmids into E. coli.• Isolated plasmids from E. coli:

A. Sequencing and BLAST searches were used to identify the proteins. B. Library plasmids were cotransformed with pGBKT7-Lhx2 back into yeast to

confirm the interactions.

Preparing Screened Plasmids For Sequencing

Clone # Protein Sequence Score #ID

11.2 Hemoglobin, beta adult s chain (Hbb-bs) 974 AC000029

13.7 Rho-related BTB domain containing 3 (Rhobtb3), X3 675 XM006517412

16.8 Actin, gamma, cytoplasmic 1 (Actg1), 1664 NM009609

18.4 Ribosomal protein L18A (Rpl18a) 503 NM029751

19.4 DEAD box polypeptide 50 (Ddx50), X2 207 XM006514346

DEAD box polypeptide 50 (Ddx50), X1 207 XM006514345

23.5 Downstream of Stk11 (Dos), X14 1214 XM006512978

25.2 microRNA 137 (Mir137) 87.9 NR029551

28.1 Ribosomal protein S10 (RPS10), X3 374 NM001204091

29.1 microRNA 137 (Mir137) 102 NR029551

29.6 Downstream of Stk11 (Dos), X14 1214 XM006512978

37.1 Quiescin Q6 sulfhydryl oxidase 2 (Qsox2) 381 XM006497902

43.1 Exoribonuclease 3 (Eri3), X4 1762 NM001285902

43.3 ADP-ribosylarginine hydrolase (Adprh) 438 NM007414

50.8 Mus musculus enolase 1, alpha non-neuron (Eno1), X3 1535 XM006538527

55.1 p21 protein (Cdc42/Rac)-activated kinase 3 (Pak3), X8 1088 XM006528754

55.3 Tubulin, beta 3 class III (Tubb3) 287 NM 023279

Plasmids Extracted and Confirmed

225 colonies pulled from screen

201 after -4 plating

127 after E coli transformation

25 sequenced successful and matched with proteins

Deadbox Ddx50

• Asp-Glu-Ala-Asp (DEAD)• RNA helicases • 38 human deadbox proteins• Alters secondary structures of RNAs. • Regulates embryogenesis, spermatogenesis,

and cellular growth and division.

Lhx2 Expression at E14.5Visel A, (2004) Nucleic Acids Res. 1;32:D552-6

Ddx50 Expression at E14.5Diez-Roux, G., et al. (2011) PLoS Biol. 9(1).

• Enhanced RNAi 3’ mRNA Exonuclease.• Exonucleases are enzymes that cleave nucleotides one at a time

from the 3’ end of a polynucleotide chain by hydrolyzing phosphodiester bonds.

• May degrade mRNA to repress translation.

Eri3

Lane 9-11: pGADT7-Ddx50Lane 12-14: pGBKT7-Ddx50Lane 15: Ladder

Lane 3-5: pGADT7-Eri3Lane 6-8: pGBKT7-Eri3Lane 15: Ladder

Eri3: .5 kBDdx50 3 KBpGBKT7: 7.3 KBpGADT7: 8.0 KB

• Lane 4 shows Eri3 and pGADT7

• Lane 7 shows Eri3 and pGBKT7

• Lane 10, 11 shows Ddx50 and pGADT7

• Lane 12-14 shows Ddx50 and pGBKT7 - 8 - 8

- 3 - 3

- .5 - .5

Cloning Full Length Open Reading Frames for Eri3 and Ddx50

Ddx50-Lhx2

• Clone 19-4 matches Ddx50 version 0 and 1

• Clone of Ddx50 matches version 2

• Clone 19-4 not included in Ddx50 version 2

Ddx50-Lhx2

Spot Plate Assays Confirm the Interaction Between Eri3 and Lhx2

LIM Domain Homeodomain

LIM Domain

No Homeodomain

No LIM Domain

Homeodomain

0 Amino Acid

180Amino Acid

255 Amino Acid

406 Amino Acid

Eri3-Lhx2 Homeodomain

LIM Domain Homeodomain

LIM Domain

No Homeodomain

No LIM Domain

Homeodomain

0 Amino Acid

180Amino Acid

255 Amino Acid

406 Amino Acid

Eri3-Lhx2 No Lim domain

LIM Domain Homeodomain

LIM Domain

No Homeodomain

No LIM Domain

Homeodomain

0 Amino Acid

180Amino Acid

255 Amino Acid

406 Amino Acid

Eri3-Lhx2 No Homeodomain

LIM Domain Homeodomain

LIM Domain

No Homeodomain

No LIM Domain

Homeodomain

0 Amino Acid

180Amino Acid

255 Amino Acid

406 Amino Acid

This suggests that the LIM domain of Lhx2 is sufficient and necessary for binding to Eri3.

Eri3-Lhx2 Lim domain

Conclusions and Future Work

Transcription:• Binding of Ddx50 or Eri3 to Lhx2 could

modulate which genes Lhx2 turns on and off.

Possible transcriptional functions for the Lhx2-Ddx50 and Lhx2-Eri3 interactions

Possible Posttranscriptional Lhx2-Ddx50 Function

• Lhx2 could recruit RNA to Ddx50:• Remove proteins from RNA

• Allows the mRNA to be translated• Allows the mRNA to be degraded

• Allow for proper folding of mRNA to allow transcription

Lhx2 binds mRNA

Lhx2 recruits Ddx50 to mRNA

Ddx50 unfolds mRNA

Possible posttranscriptional Lhx2-Eri3 function

Post transcription:• Lhx2 could inhibit the Eri3 for

degrading mRNA• Lhx2 could recruit RNA to Eri3:

• Degradation of mRNA

Lhx2 interacts with mRNA

Lhx2 recruits Eri3

Eri3 degrades mRNA

Potential Pathway

• Lhx2 may interact with both Eri3 and Ddx50.

• Lhx2 may recruit mRNA. • Ddx50 unfolds mRNA and allows Eri3 to

degrade the protein.• The mRNA that are destroyed could be

transcription factors that repress formation of the visual area.

• Determining the expression pattern of Eri3 will help to refine these models.

Lhx2 interacts with folded mRNA Ddx50 and Eri3 get recruited by Lhx2

Ddx50 unfolds mRNA

Eri3 is able to degrade the mRNA now that the mRNA is unfolded

• Need to clone the full length Ddx50, to confirm that it interacts with Lhx2.

• Compare expression of Ddx50 and Eri3 to Lhx2 in neocortex using in situ hybridization.

• Yeast 3 hybrid

• RNA experiments:• Determine whether Lhx2 interacts with

mRNA.• Functional assay will detect whether Lhx2

affects the Eri3-dependent degradation of mRNA.

Future Research

• Dr. Kroll• Thesis Committee

• Dr. Carin Thomas• Dr. Holly Pinkart

• Dr. Kroll’s research group• Family And Friends• CWU

Acknowledgements

• Montagnini, A.; Treves, A. The evolution of mammalian cortex, from lamination to arealization. Brain Res Bull 2003, 60 (4), 387-393.

• O'Leary, D. D.; Chou, S. J.; Sahara, S. Area patterning of the mammalian cortex. Neuron 2007, 252-269.• Tétreault, N.; Champagne, M. P.; Bernier, G. The LIM homeobox transcription factor Lhx2 is required to specify

the retina field and synergistically cooperates with Pax6 for Six6 trans-activation. Dev Biol 2009, 541-550.• Matchmaker® Gold Yeast Two-Hybrid System User Manual; ClonTech Laboratories, Inc: Mountain View, CA.• ERI1 Exoribonuclease Family Member 3. http://www.genecards.org/cgibin/carddisp.pl?

gene=ERI3&search=e268f81180ef17c582e4d36fc56ae1db (accessed Dec 12, 2014).• Dubnau, J.; Struhl, G. RNA recognition and translational regulation by a homeodomain protein. Nature 1996, 379

(1), 694-699.• Takashi, K.; Masaya, O.; Takahiko, U.; Shoko, K.; Kohji, O.; Toshihiko, E.; Fumihiro, F.; Yasufumi, M. Dbp9p, a

Member of the DEAD Box Protein Family, Exhibits DNA Helicase Activity. The Journal of Biological Chemistry 2004, 279 (20), 20692-20698.

• B, S.; Hari, P.; Huilgol, D.; Tole, S. Dual role for LIM-homeodomain gene Lhx2 in the formation of the lateral olfactory tract. The Journal of Neuroscience 2007, 27 (9), 2290-2297.

• Susa, T.; Sato, T.; Ono, T.; Kato, T.; Kato, Y. Cofactor CLIM2 promotes the repressive action of LIM homeodomain transcription factor Lhx2 in the expression of porcine pituitary glycoprotein hormone alpha subunit gene. Biochimica et Biophysica Acta 2006, 1759 (8), 403-409.

Citations

Complex with RNA and Protein

Lhx2-Ddx50 with RNA and Protein

RNA Interference

RIP Assay

Area migrations after knockout of other neocortical proteins

Co-IP