INTRODUCTION/BACKGROUND

Our lab is interested in the genetics behind placental development, and is specifically interested in trophoblast invasion. Trophoblast invasion is very important during placental development, because it establishes blood flow between the mother and fetus. There are many transcription factors that are involved in the regulation of trophoblast invasion, and one of the most prominent in this process is Hypoxia-Inducible-Factor(HIF). HIF is activated in low oxygen environments and is responsible for the transcriptional upregulation of genes important for angiogenesis and cell invasion. Furthermore, HIF mouse knockout models have defects in placental development showing HIF is a key factor in healthy placental and fetal development. One of our main objectives is to map the thousands of potential HIF binding sites during trophoblast invasion in mouse using ChIP-Seq. Our lab has already done HIF ChIP experiments and obtained target enrichment that was promising, but not optimal. The objective of this project was to optimize the antibody binding time during the ChIP protocol with a main goal of increasing HIF target enrichment, so that a genomic library of its targets could be made.

The material presented here is based upon work supported by the National Science Foundation under Award No. EEC-0813570. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

Optimizing ChIP in Mouse Placenta Divyesh Kumar, Anilisa Biswas, and Geetu Tuteja, Department of Genetics, Development, and Cell Biology, Iowa State University

RESEARCH QUESTION/HYPOTHESIS

How does increasing antibody binding time to transcription factors or histones affect qPCR enrichments?

Our hypothesis is that the longer the antibodies are allowed to bind to the chromatin, the level of enrichments/fold change would be higher after qPCR.

REFERENCES

1. Esopi, David. "A Starter Chromatin Immunoprecipitation (ChIP) Protocol." What Is Epigenetics. What Is Epigenetics?, 10 Feb. 2015. Web. 24 July 2016.

2. Schmittgen, Thomas D., and Kenneth J. Livak. "Analyzing real-time PCR data by

the comparative CT method." Nature protocols 3.6 (2008): 1101-1108.

RESULTS

METHODS

DISCUSSION The graphs show that the highest enrichments are obtained when antibodies are

given a longer period of time to bind to their targets (2:00 pm and 3:00 pm). Using this data, the next step would be setting up a HIF ChIP experiment with the optimized antibody binding time to see if higher levels of enrichments are obtained. If the enrichments from the HIF ChIP are significant, then a ChIP-Seq library can be generated. If the enrichments from the HIF experiment are not significant, more experiments can be done on the optimization of ChIP in different parts of the protocol and not necessarily in the antibody binding stage of the ChIP protocol.

Figure 1. Chromatin Shearing Figure 2. HIF-1a Enrichments

Figure 3. H3K27Ac (11am, 1pm, 3pm) Figure 4. H3K27Ac (9am, 10am, 11am, 12pm, 1pm, 2pm)

•  Columns 1 and 2 show implantation site shearing. •  Columns 3 and 4 show placenta shearing.

•  This graph shows promising but not optimal HIF ChIP enrichments.

•  High enrichments are shown for antibody binding until 3pm. •  High enrichments are shown for antibody binding until 2pm.

1 2 3 4

ACKNOWLEDGEMENTS I would like to thank Dr. Geetu Tuteja, Anilisa Biswas, Bekah Starks, Ashish Jain,

David Robbins, Elizabeth Melton, Amanda Loke, and CBIRC for making my research experience possible.