The TCHP method functions as an advanced targeted chromatin purification system allowing scientists to selectively extract particular chromatin regions from cell nuclei with their connected protein and RNA complexes remaining intact.
Targeted chromatin purification (TCHP) applies a double pull-down technique where a tetracycline-responsive "hook" attaches to a specific promoter. The suppression of multiple transcription factors in human primary erythrocytes causes the activation of γ-globin gene expression. Therapeutic approaches for β-thalassemia and sickle cell disease may target the factors responsible for γ-globin gene expression reactivation which alleviates patient symptoms. Proteomics technology sensitivity keeps advancing enabling scientists to isolate tiny protein quantities which they can then identify through mass spectrometry.
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Principle of Targeted Chromatin Purification (TCHP) Method
TCHP targets the target locus through a specific guide RNA (sgRNA) and is purified by binding affinity tags (such as Biotin, FLAG, GFP, etc.). The main steps are as follows:
| Construct dCas9-SunTag complex |
By binding multiple SunTags (tandemly repeated antibody epitopes) through dCas9 to increase affinity. |
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Binding GFP or other reporter tags to improve visualization. |
| Design specific sgRNA |
Select sgRNA targeting the target chromatin region (such as the γ-globin locus). |
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By optimizing the sgRNA sequence, the binding efficiency is improved. |
| Express dCas9-SunTag and related proteins |
Co-express dCas9-SunTag and antibody fusion proteins (such as scFv-GFP, scFv-Biotin) in cells. |
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Affinity purification by GFP or Biotin. |
| Chromatin cross-linking and shearing |
1% formaldehyde cross-linking fixes chromatin complexes. |
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Chromatin is sheared to 200–500 bp fragments by sonication or enzymatic digestion. |
| Affinity purification (Pull-down) |
Immunoprecipitation is performed using Streptavidin beads or anti-FLAG/GFP beads. |
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Stringent washing removes non-specific binders. |
| Downstream analysis |
Proteomics (LC-MS/MS): Analyze chromatin-bound protein complexes.RNA-seq: Analyze RNA binding to targeted chromatin regions. |
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ATAC-seq / ChIP-seq: Study chromatin accessibility and histone modification status. |
Advantages of Targeted Chromatin Purification (TCHP)
- Efficient enrichment: Compared with ChIP, which relies on antibody recognition, TCHP directly targets DNA, which has higher enrichment efficiency.
- Compatible with multiple analysis techniques: It can be combined with multi-omics methods such as proteomics, RNA-seq, and ChIP-seq.
Purification of Repressed γ-globin Chromatin Centers
The erythroid progenitor cell line was derived from LCR-TetO-γ::TetR3T::BirA::p53null fetal liver at E13.5. The cell line was cloned and further expanded in vitro. Purification of repressed γ-globin chromatin centers typically involves chromatin immunoprecipitation (ChIP), affinity purification, or other chromatin isolation techniques. Here is an overview of a common experimental procedure:
Cell Culture and Treatment
Select a suitable cell line from options like human erythroid progenitor cells (for instance, K562 cells) or primary hematopoietic cells. Cells are grown in suitable conditions and the usage of specific compounds like hydroxyurea or HDAC inhibitors enables modulation of γ-globin expression.
Chromatin Fixation
Cells are cross-linked using 1% formaldehyde to fix protein-DNA complexes, typically for 10 minutes at room temperature. Cross-linking is terminated (e.g., by adding 125 mM glycine for 5-10 minutes).
Chromatin Shearing
Chromatin is sheared to 200-500 BP fragments by sonic Ation or enzymatic digestion. optimize sonic Ation conditions to avoid excessive degradation.
Washing and Elution
Wash with low salt, high salt, lithium salt, and TE buffer sequentially to remove nonspecific binders. Elute the DNA-protein complex by heating or chemical methods.
DNA Analysis
Analyze the chromatin state of the γ-globin repressor region by qPCR or sequencing (ChIP-seq).
Proteomic Analysis of Chromatin Pull-downs
The chromatin pull-down proteomics technique allows researchers to detect proteins linked to distinct chromatin areas or changes in chromatin modification states. This approach merges chromatin immunoprecipitation (ChIP) or affinity purification methods with mass spectrometry (MS) analysis to determine which proteins associate with chromatin. It is performed through the following steps:
- Cell culture and treatment
- Chromatin fixation
- Cell lysis and chromatin isolation
- Chromatin pull-down experiment
- Protein elution and digestion
- Mass spectrometry analysis (LC-MS/MS)
- Data analysis
TCHP: an Efficient Detection Method
The TCHP system creates a powerful chromatin purification framework which enables researchers to examine specific loci proteomes and RNA binding proteins along with chromatin modifications. The method shows extensive application capabilities for epigenetic studies and disease research including β-thalassemia as well as drug screening purposes.
The TCHP approach enables scientists to pinpoint chromatin factors associated with specific target site sequences through in vivo purification. The TChP method described here demonstrates broader applicability for isolating unique sequences despite its results being non-comparable to Déjardin and Kingston's (2009) findings. Fundamental differences exist between both methods which lead to distinct pros and cons for each. The PICh approach uses a hybridization step to secure the target chromatin while TChP functions through TetR protein binding to the TetO sequence. The TChP method can outperform hybridization in efficiency yet requires an artificial sequence which functions as a "bait" to be incorporated into the natural sequence. The introduction of the bait must be validated through controls to confirm that it does not alter the target sequence's behavior. Comparable negative controls must be performed in every approach to identify candidate binding proteins.
References
- Pourfarzad, F., et al. Locus-specific proteomics by TChP: targeted chromatin purification. Cell reports. 2013, 4(3): 589-600.
- Zhukova, L., et al. INSIGHT-HER2BC: Real-world evidence of TCHP regimen to describe the best patient profile and open up questions-The interim results. 2023: 12607-12607.
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