Purification of proteins associated with specific genomic Loci

Abstract

Eukaryotic DNA is bound and interpreted by numerous protein complexes in the context of chromatin. A description of the full set of proteins that regulate specific loci is critical to understanding regulation. Here, we describe a protocol called proteomics of isolated chromatin segments (PICh) that addresses this issue. PICh uses a specific nucleic acid probe to isolate genomic DNA with its associated proteins in sufficient quantity and purity to allow identification of the bound proteins. Purification of human telomeric chromatin using PICh identified the majority of known telomeric factors and uncovered a large number of novel associations. We compared proteins found at telomeres maintained by the alternative lengthening of telomeres (ALT) pathway to proteins bound at telomeres maintained by telomerase. We identified and validated several proteins, including orphan nuclear receptors, that specifically bind to ALT telomeres, establishing PICh as a useful tool for characterizing chromatin composition.

Figure 1

Outline of the PICh Protocol

Figure 2. Purification of Telomeric Chromatin from Transformed Human Cell Lines

(A) Silver staining of material obtained from PICh purified telomere chromatin. Purifications from the two HeLa clones (S3 and 1.2.11) and the WI38-VA13 ALT cell line are shown. T: PICh performed with the telomere-specific probe. S: PICh performed with the “scrambled” probe. Input represents 0.001% of the starting material (3 × 10⁴ cell equivalent), and purifications were from 5 × 10⁸ cell equivalents/lane. (B) Validation of selected PICh associations to ALT telomeres by immunostaining. Protein names are followed by their ranking order in the ALT list. NXP2 was an HA-tagged construct transiently transfected (experiment performed 48 hr post-transfection). Endogenous RIP140 and Fanc-J are detected using specific antibodies. (C) Coimmunostaining with the Flag-tagged HMBOX1 and RAP1 in the WI38-VA13 and HeLa 1.2.11 cell lines (72 hr post-transfection). (B and C) Quantification on the right is expressed as the fraction of nuclei showing the extent of telomere colocalization with the tested proteins in the analyzed population.

Figure 3. Analysis of COUP-TF2, COUP-TF1, and TR4 Association with WI38-VA13 ALT Telomeres

(A) CHIP with anti-COUP-TF2 and anti-TR4 antibodies. Immunoprecipitated DNA (from VA13 or HeLa 1.2.11) was probed on a slot-blot using a telomere-specific probe (Telomere) or an Alu probe (Alu). Inputs represent 0.002% of starting material. Twenty percent of the IP was loaded. The right panel shows quantification performed from two independent experiments (error bars represent SD from enrichment values). Immunoblots for COUP-TF2 and TR4 on PICh-purified telomeres (6% loaded) are shown. (B) Western blot analysis for COUP-TF2 and TR4 levels in material purified from the indicated cell lines; TRF2 is a loading control for telomere material. (C) Immunoblots showing COUP-TF2, COUP-TF1, and TR4 protein levels in HeLa 1.2.11 or VA13 cell lysates. β-tubulin is probed to control for loading. (D) Immuno-FISH (COUP-TF2 and telomere probe) and coimmunostaining of RAP1 and TR4 or COUP-TF1 in VA13 nuclei. Frequency of colocalization is shown on the right.

Figure 4. Coimmunostaining of Orphan Receptors with Shelterin Proteins on VA13 Metaphase Spreads

Top panels: COUP-TF2 and TRF2. Bottom panels: TR4 and RAP1.

Figure 5. Nucleosomal Density and Composition at Telomeres

(A) similar amounts of shelterins from HeLa 1.2.11 or S3 were loaded to compare nucleosomal histone to shelterin signals. (B) CHIP with an anti-H3 antibody. The immunoprecipitated DNA (from HeLa S3 or 1.2.11) was probed on a slot-blot using a telomere-specific probe (Telomere) or an Alu probe (Alu); the panel on the right shows quantitation from two independent experiments. Inputs represent 0.002% of starting material. Twenty percent of the IP was loaded. Right panel shows quantifications normalized to input telomere DNA and H3 IP at Alu repeats (error bars represent SD from enrichment values). (C) CHIP with anti-H1 and anti-H3 antibodies. Immunoprecipitated DNA (from VA13 or HeLa 1.2.11) was probed on a slot-blot using a telomere-specific probe (Telomere) or an Alu probe (Alu); the panel on the right shows quantification from two independent experiments (error bars represent SD from enrichment values). Inputs represent 0.002% of starting material. Twenty percent of the IP was loaded. H1 versus H3 enrichments at telomeres are normalized using the signals obtained for these proteins with the Alu repeats.

Figure 6. Possible Scenario for Orphan Receptor Binding to ALT Telomeres

R: purine (A or G), DR: direct repeat. A combination of telomere variant repeat may constitute binding sites for orphan receptors, which upon association to telomeres target the locus to PML-NB.