Chromatin immunoprecipitation assays for Myc and N-Myc

Abstract

Myc and N-Myc have widespread impacts on the chromatin state within cells, both in a gene-specific and genome-wide manner. Our laboratory uses functional genomic methods including chromatin immunoprecipitation (ChIP), ChIP-chip, and, more recently, ChIP-seq to analyze the binding and genomic location of Myc. In this chapter, we describe an effective ChIP protocol using specific validated antibodies to Myc and N-Myc. We discuss the application of this protocol to several types of stem and cancer cells, with a focus on aspects of sample preparation prior to library preparation that are critical for successful Myc ChIP assays. Key variables are discussed and include the starting quantity of cells or tissue, lysis and sonication conditions, the quantity and quality of antibody used, and the identification of reliable target genes for ChIP validation.

Fig. 1

Schematic diagram illustrating major steps in the Myc ChIP process

Fig. 2

Agarose gel electrophoresis demonstrating appropriate chromatin size for ChIP. This chromatin check is performed prior to IP (see step 3.2, item 9) and can be used to fine-tune the sonication time. The left panel shows chromatin fragments that are too large; additional sonication cycles should be performed before continuing. The right panel shows chromatin fragments of the optimal size range. An additional, more accurate chromatin size check can be performed after the IP, using leftover supernatant from the IP (see step 3.3, item 6)

Fig. 3

Representative results showing end-point PCR validation of ChIP assays in human cells using APEX1 primers given in Table 4 (see step 3.5, item 2). (a) ChIP in Tet21N neuroblastoma cells. Top panel, cells that overexpress N-Myc; bottom panel, cells after 3 days of tetracycline treatment which blocks N-Myc expression. (b) ChIP for both c-Myc and N-Myc in hESCs (two 6-well plates of cells per IP). Antibodies: N-Myc (Santa Cruz, sc-53993), c-Myc (Abcam, ab56), H3K9ac (Abcam, ab12179), and H3K4me3 (Millipore, 04–745)