Regulation of chromatin structure and gene expression is essential for normal development and cellular growth, and gene transcription involves tightly controlled protein:DNA interactions. Currently there is a rapidly growing trend towards genome-wide identification of protein binding sites on chromatin and characterization of the transcriptome using the chromatin immunoprecipitation (ChIP) method coupled with either DNA microarray or ultrahigh-throughput sequencing analysis. While significant advances and the development of new technology in the fields of DNA microarrays and sequencing have allowed genomic-level characterization of protein binding sites, the upstream ChIP process for the capture of DNA fragments remains cumbersome and time consuming.
The HaloCHIP™ System uses a protein tag, the HaloTag® moiety, which can be fused to any protein and which mediates a covalent interaction with various interchangeable ligands. In the HaloCHIP™ System, DNA binding proteins fused to the HaloTag® moiety can be transiently or stably expressed in cells. Similar to the ChIP protocol, cells are treated with formaldehyde, generating covalent protein:DNA crosslinks, lysed, and sonicated to shear chromatin into 500–1500 bp fragments. Unique to the HaloCHIP™ System, the crosslinked complexes are then directly and covalently captured from cell lysates on HaloLink™ Resin and washed stringently to remove nonspecific proteins and DNA. Captured DNA fragments are released from the HaloLink™ Resin using heat, which reverses the formaldehyde crosslinks. The DNA can be purified using a PCR purification kit and analyzed by the researcher’s preferred method.
The entire process—from crosslinking to DNA release—can be accomplished in 24 hours using the HaloCHIP™ System, compared to five days for standard ChiP.
A paper by Hartzell et al. analyzes the CREB signaling pathway by using the HaloCHIP™ System to map the genome-wide targets of the CREB protein:
Hartzell, D. et al. (2009) A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high-throughput reporter assays. BMC Genomics 10, 497.
And two publications have used the HaloCHIP™ System to identify transcriptional regulators:
Du, X., Rosenfield, R.L, and Qin, K. (2009) KLF15 is a transcriptional regulator of human 17 beta-Hydroxysteroid Dehydrogenase Type 5 Gene. A potential link between regulation of testosterone production and fat stores in women. J. Clin. Endocrinol. Metab. 94, 2594–601.
Jag, U.R., Zavadil, J., and Stanley, F.M. (2009) Insulin acts through FOX03a to activate transcription of plasminogen activator inhibitor type 1. Molecular Endocrinology 23, 1587–602.
Latest posts by Michele Arduengo (see all)
- Control Samples: Three Terrifying Tales for Scientists - October 31, 2018
- Forensic Scientists Improve Sexual Assault Kit Turnaround Time with Y-Screening - July 27, 2018
- Shaping the Future by Investing in Science - July 19, 2018