The Role of Krüppel-Like Factor 2 in Erythroid Cells

1. The Role of Krüppel-Like Factor 2 in Erythroid Cells Bryan Castro Joyce Lloyd (Mentor ) Virginia Commonwealth University Dept of Human and Molecular Genetics

2. Background Information Sickle Cell Anemia Hemoglobin KLF2 β-Globin Promoter Cre Recombinase

3. Sickle Cell Anemia • Hemoglobin that sickles • Inherited Hemoglobinopathy • Predominant in people of African or Mediterranean ancestry

4. Sickle Cell Anemia Genetic mutation of an Adenine to Thymine Protein mutation of a hydrophilic amino acid to a hydrophobic one Noguchi

5. Globin Switching Schechter, 2008

6. Hemoglobin • Sickle-Cell Anemia is caused by a mutation in the adult β chains of the hemoglobin structure • Thalassemia is a reduced production of α/β globin King, 2009 Can embryonic globins be turned on to take place of dysfunctional adult globins?

7. Krüppel-Like Factor 2, KLF2, is a transcription factor important in gene expression during development and differentiation. Vasculature Lungs Erythroid morphology and function KLF2 * Transcription factors are proteins that initiate or regulate the process of transcription, in which RNA is created from DNA (Campbell and Reece, 2004)

8. KLF2 -/- Embryonic Day 12.5 (E 12.5) KLF2 null embryos have a yolk sac that lacks blood They also show growth retardation as compared to wildtype Wani et al., 1998

9. KLF2 and Human ε KLF2 plays part in the expression of the human embryonic gene εin transgenic mice KLF2 might have the therapeutic value in treating heminoglobinopathies Basu et al., 2005

10. The β-globin locus contains CACCC binding sites in the promoters of the β-like genes, which could serve as targets for KLF2 binding Erythroid Krüppel-Like Factor, EKLF, binds the β-globin genes through their CACCC motif EKLF and KLF2 have high similarities in their zinc fingers which means that KLF2 might be able to bind to this CACCC element β-Globin Promoters Knight and Shimeld, 2001

11. This enzyme eliminates targeted sequences by binding to both of the loxP sites and bringing them together to remove the unwanted exon Cre Recombinase • Cre can be used under the control of tissue-specific promoters, deleting genes only in those cells • Mice with this construct were used in the study to conditionally knockout the KLF2 in erythroid cells Rosenthal and Brown, 2007

12. Methods and Materials Mating Genotyping KLF2 mRNA Quantification

13. Mating KLF2 F/+, βCre KLF2 F/F KLF2 F/F, βCre KLF2 F/+, βCre KLF2 F/F KLF2 F/+

14. Experiments • Polymerase Chain Reactions (PCR) To amplify wanted DNA sequences • Quantitative Reverse Transcriptase-PCR To check if there is KLF2 mRNA reduction Pitocchelli, 2001 Phenol-Chloroform Extraction RNA Isolation

15. Results and Conclusion Genotyping Results KLF2 mRNA Reduction? What now?

16. Genotyping Sample Cre Picture If βCre is present then a single band will appear, band size: 256 base pairs (bp) + - 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Sample Flox Picture F/F +/+ F/+ Upper band- +/+, 376 bp Lower band- F/F, ~303 bp Both bands- F/+ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

17. Percent KLF2 mRNA in E 10.5 blood Sample #595 can be used for the experiment since it shows significant reduction of KLF2 mRNA At least 2 more samples are needed Courtesy of Mohua Basu

18. Data Interpretations • If embryonic globin mRNA is: • Reduced, this would suggest that KLF2 may directly regulate embryonic globin gene expression by binding to the CACCC motif • Not reduced, KLF2 might play an indirect role in the expression of embryonic globin genes in erythroid cells

19. Acknowledgements National Institutes of Health- NIDDK Virginia Commonwealth University Dr. Joyce Lloyd Mohua Basu and other Lloyd lab members Dr. Suzanne Barbour Dr. Carolyn Conway Maura Murphy Jerry Lingrel, University of Cincinnati (KLF2 F/F mice) Kenneth Peterson, University of Kansas (βCre mice)

20. Questions?