Dual agarose magnetic (DAM) ChIP

Abstract

Background: Chromatin immunoprecipitation (ChIP) has become a very popular technique to study epigenetic regulation because it can be used to identify proteins and protein modifications present at specific locations in chromatin. While techniques have been developed to investigate epigenetic modifications present in chromatin during a specific biological function such as transcription, they depend upon the ability of the ChIP to analyze two epitopes on the same chromatin and are generally time consuming, difficult to perform, and not very sensitive. The Dual Agarose Magnetic (DAM) ChIP procedure described here is designed to address these shortcomings.

Findings: Protein A agarose and protein G magnetic beads bound with different IgGs have been combined in a single Chromatin Immunoprecipitation (ChIP) assay to analyze for the presence of two epitopes on the same chromatin at the same time. This procedure has been used with non-immune rabbit IgG bound to either the agarose or beads in order to include an internal negative control for non-specific binding of chromatin. The procedure has also been used with various antibodies including those targeting RNA Polymerase II and replication protein A 70 to determine whether specific forms of modified histones are present in either transcribing or replicating forms of SV40 minichromosomes respectively.

Conclusions: The DAM ChIP procedure is a rapid, simple, and sensitive technique to characterize two epitopes located in the same chromatin. It should be particularly useful for the rapid screening of epigenetic modifications present in biologically active chromatin.

Figure 1

Schematic for Dual Agarose Magnetic (DAM) ChIP protocol.

Figure 2

DAM ChIPs. 48 hour unfixed SV40 minichromosomes were analyzed by (a) standard (Std ChIP) or (b, c, d) DAM chromatin immunoprecipitation (DAM ChIP) and the immunoprecipitates were amplified by PCR (32 cycles) with primer sets to the early region of the SV40 genome as previously described [5-7]. (a) Std ChIPs with antibodies bound to protein A agarose or protein G magnetic beads. Lane 1, Std ChIP with IgG (7.5 μl) (Bethyl Laboratories; P120-101) bound to agarose (Millipore, 17-295); lane 2, Std ChIP with Hyp H4 (7.5 μl) (Millipore; 06-866) bound to agarose; lane 3, Std ChIP with IgG (7.5 μl) bound to magnetic beads (Active Motif, 53014); lane 4, Std ChIP with Hyp H4 (7.5 μl) bound to magnetic beads. Ag; Agarose, MB; magnetic beads. (b) DAM ChIPs in which chromatin was analyzed with IgG (7.5 μl) bound to agarose (Lane 1) and Hyp H4 (7.5 μl) bound to magnetic beads (Lane 2) in the same tube or IgG (7.5 μl) bound to magnetic beads (Lane 3) and Hyp H4 (7.5 μl) bound to agarose (Lane 4) in the same tube. (c) DAM ChIPs immune selected with antibody to Hyp H4 bound to magnetic beads. Purified immune selected chromatin was divided into two aliquots and an aliquot was added to a tube containing either IgG or Hyp H3 bound to agarose. Lane 1, DAM ChIP with IgG (7.5 μl); lane 2, DAM ChIP with Hyp H3 (7.5 μl) (Millipore; 06-599). DAM ChIPs immune selected with antibody to Hyp H4 bound to agarose. Purified immune selected chromatin was divided into two aliquots and an aliquot was added to a tube containing either IgG or Hyp H3 bound to magnetic beads. Lane 1, DAM ChIP with IgG (7.5 μl); lane 2, DAM ChIP with Hyp H3 (7.5 μl).

Figure 3

Schematic representation of the strategy used for DAM ChIP analysis of protein modifications in immune selected SV40 minichromosomes.

Figure 4

DAM ChIP analysis of replicating and transcribing SV40 minichromosomes. 48 hour unfixed SV40 minichromosomes were analyzed by [a] standard chromatin immunoprecipitation using either IgG, or antibodies to H3K9me1, H3K9me2, H3K9me3, or Hyp H4 (7.5 μl each) bound to protein A agarose or [b] DAM ChIPs using antibody bound to magnetic beads in the first step which recognized RPA70 (SCBT; sc-25376) (50 μl) to immune select replicating minichromosomes or antibody which recognized RNAPII (SCBT; sc-900) (50 μl) to immune select transcribing minichromosomes. Following immune selection the magnetic beads were washed according to protocol and then divided into five equal aliqouts with an aliquot added to a tube which contained either IgG, or antibodies to H3K9me1, H3K9me2, H3K9me3, or Hyp H4 (7.5 μl each) bound to agarose. The immunoprecipitates bound to agarose from both analyses were then amplified by PCR (a: 32 cycles or b: 45 cycles) with primer sets to the early region of the SV40 genome as previously described [5-7]. Lane 1, ChIP with IgG; lane 2, ChIP with H3K9me1 (Abcam; ab9045); lane 3, ChIP with H3K9me2 (Abcam; ab1220); lane 4, ChIP with H3K9me3 (Abcam; ab8898); lane 5, ChIP with Hyp H4.