Gene Regulation by Aebp2Arundhati Bakshi, Hana Kim, Joomyeong Kim

Louisiana State University

Abstract

Conclusion

References

1. Kim H, Kang K, Kim J. AEBP2 as a potential targeting protein for Polycomb Repression Complex PRC2. Nucleic Acids Res. 2009 May;37(9):2940-50.

2. Paquette J, Tokuyasu T. EGAN: exploratory gene association networks. Bioinformatics. 2010 Jan 15;26(2):285-6.

Acknowledgements

The authors would like to thank the entire Kimlab, especially Dr. Michelle Thiaville and Muhammad Ekram and our funding agency, Howard Hughes Medical Institute.

1. Cross-link chromatin

2. Random shearing by sonication

3. Immunoprecipitation with Aebp2-specific antibody

4. Restriction Enzyme Digestion

5. Reverse Crosslink and cleanup

6. Sequence and map to genome

Fig. 2. Chromatin Immunoprecipitation ReactionChromatin Immunoprecipitation-Sequencing assay was done on an adult mouse brain to test the loci at which Aebp2 binds in the mouse genome. Genes up to 5000 base pairs from the ChIP-Seq peaks were evaluated using the bioinformatics software EGAN (Exploratory Gene Association Network).

Figure 3. Gene Association Network controlled by Aebp2The output from EGAN shows a network of genes potentially controlled by Aebp2. It suggests that Aebp2 may regulate genes involved in the development of the central nervous system and the generation and development of neurons in the embryonic stage. Relationships between the genes that Aebp2 binds to are also portrayed in the figure (legend inset).

Pubmed co-occurrenceHuman protein-protein interactionProximity on the chromosome

• Aebp2 is highly expressed along the spinal cord of the mouse embryo, the dorsal root ganglion and in cells derived from the neural crest cells.• A gene association network potentially controlled by Aebp2 (generated using EGAN) suggests that it may regulate genes involved in central nervous system development and neurogenesis, especially in the embryonic stage.• RT Primers were developed and tested positive for Mecp2, Ctnna2, Tbr1 and Atp2b2 on cDNA derived from mouse embryo and adult thymus. • These primers will be used in the future to perform a Q-RT PCR to test differential gene expression in Aebp2 WT versus mutant mice.• The ChIP assay will also be repeated to verify that these loci indeed bind to Aebp2.

Aebp2 is an evolutionarily well-conserved gene whose protein binds to DNA and is potentially involved in embryonic development and gene regulation.1 In this study, we performed a beta-galactosidase stain to identify the expression pattern of Aebp2 on a 15.5 day old mouse embryo. Aebp2 appeared to be highly expressed along the spinal cord, the dorsal root ganglion and in cells derived from the neural crest cells. Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) data from an adult mouse brain was analyzed for genes up to 5000 base pairs from the peaks and evaluated using the bioinformatics software EGAN.2 The output showed a network of associated genes involved in central nervous system development and neurogenesis, potentially controlled by Aebp2. Reverse Transcription (RT) primers were positively tested for Mecp2, Ctnna2, Tbr1 and Atp2b2 in Aebp2 wild-type (WT) mouse embryo and adult thymus cDNA. Future analysis would include performing a Quantitative Real-Time (Q-RT) PCR on cDNA from mRNA extracted from adult brain as well as embryo and repeating the ChIP assay for reproducibility. 

A B

Figure 1. Beta-Gal Staining of 15.5 days old mouse embryo(A) shows the transverse section of the embryo with high expression rate of Aebp2 around the neural tube. (B) shows the saggital section of the embryo. Aebp2 can be seen to be expressed highly around the dorsal root ganglion and in the cranial neural crest cells

Figure 4. RT Primer Check for genes that are potentially bound by Aebp2(A) shows the positive expression of Mecp2, Tbr1, Atp2b2 and Ctnna2 in an Aebp2 wild-type mouse embryo. (B) shows the positive expression of Mecp2 and Ctnna2 and the negative expression pattern of Tbr1 and Atp2b2 in an adult WT mouse thymus.

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