Upstream Regulator Analysis

Upstream Regulator Analysis

Identify transcription factors (TFs) driving observed differential expression by integrating ChIP-Atlas TF binding data (epigenomics) with RNA-seq DE results (transcriptomics). Ranks TFs by a combined regulatory score incorporating binding enrichment, target-DE overlap (Fisher's exact test), and directional concordance (activator vs repressor).

When to Use This Skill

Use when you:

• Have DE results and want to identify TFs driving expression changes
• Need to go beyond simple gene list enrichment to mechanistic TF-level evidence
• Want to distinguish activators (targets upregulated) from repressors (targets downregulated)
• Want to integrate epigenomics (ChIP-seq) with transcriptomics (RNA-seq) in one analysis

Don't use for:

• Single-cell DE results (designed for bulk RNA-seq DE)
• Organisms not in ChIP-Atlas (see supported genomes below)
• Histone mark analysis (use chip-atlas-peak-enrichment directly)
• When you only need TF binding enrichment without target gene integration

Requires: Internet access (ChIP-Atlas API + data server). Runtime: 15-25 minutes (API polling + target gene downloads).

Installation

pip install pandas numpy scipy requests matplotlib seaborn reportlab

Sibling skill dependencies: Requires chip-atlas-peak-enrichment and chip-atlas-target-genes directories at the same level.

Inputs

• DE results CSV/TSV with columns: gene symbol, log2 fold change, adjusted p-value
  • Supports DESeq2 (log2FoldChange, padj), edgeR (logFC, FDR), limma (logFC, adj.P.Val)
  • Column names auto-detected; override with parameters if needed
• Genome: hg38, hg19, mm10, mm9, rn6, dm6, dm3, ce11, ce10, sacCer3

Outputs

Analysis objects:

• analysis_object.pkl - Complete analysis for downstream use
  • Load with: import pickle; obj = pickle.load(open('analysis_object.pkl', 'rb'))
  • Contains: regulon_scores, enrichment results, target gene data, DE data, parameters

CSV results:

• regulon_scores_all.csv - All scored TFs with regulatory score, Fisher's p-value, concordance, direction
• regulon_scores_top.csv - Top 20 TFs
• target_overlaps.csv - Per-TF target gene overlap with DE status (up/down)
• enrichment_up.csv / enrichment_down.csv - ChIP-Atlas peak enrichment results

Visualizations (PNG + SVG):

• upstream_regulators_top_regulators - Bar chart: TFs ranked by regulatory score
• upstream_regulators_target_overlap - Stacked bar: TF targets classified as up/down/unchanged
• upstream_regulators_evidence_scatter - Scatter: ChIP enrichment vs Fisher significance
• upstream_regulators_heatmap - Clustermap: TFs × regulatory evidence metrics

Reports:

• summary_report.md - Human-readable analysis summary
• analysis_report.pdf - Publication-quality PDF with Introduction, Methods, Results, Conclusions
  • Requires: pip install reportlab (optional — markdown report generated regardless)

Clarification Questions

1. Input Files (ASK THIS FIRST):

   • Do you have DE results (CSV/TSV) to analyze?
   • If uploaded: Is this the DE results file you'd like to find upstream regulators for?
   • Expected columns: gene symbol + log2FoldChange + adjusted p-value
   • Or use example data? Three options:
     • a) Estrogen/MCF7 dataset (recommended) — real DE results from GSE51403 (estradiol-treated MCF7 breast cancer cells, ~58K genes). Expected top regulator: ESR1
     • b) Airway dataset — real DE results from GSE52778 (dexamethasone-treated airway smooth muscle cells, ~58K genes). Expected top regulator: NR3C1
     • c) Synthetic TP53-driven data (~200 genes, fast, offline)

2. Analysis Options:

   • (If using example data) Choose analysis parameters:
     • a) Standard analysis (top 10 TFs, q < 0.05) (recommended)
     • b) Comprehensive analysis (top 15 TFs, q < 0.1)
   • (If using your own data) What species/genome?
     • a) Human (hg38)
     • b) Human (hg19)
     • c) Mouse (mm10)
     • d) Other (specify)

Standard Workflow

Note: Run from the OmicsClaw root directory and add the workflow scripts to sys.path:

import sys; import os; sys.path.insert(0, os.path.abspath('knowledge_base/scripts/upstream-regulator-analysis'))

🚨 MANDATORY: USE SCRIPTS EXACTLY AS SHOWN - DO NOT WRITE INLINE CODE 🚨

Step 1 - Load data:

# For example data (real estrogen/MCF7 dataset, downloads from EBI Expression Atlas):
from load_example_data import load_example_data
de_data = load_example_data(source="estrogen")

# Alternative: airway dataset (dexamethasone, real data):
de_data = load_example_data(source="airway")

# For synthetic data (offline, fast, TP53-driven):
de_data = load_example_data(source="synthetic")

# For user data:
from load_de_results import load_de_results
de_data = load_de_results("path/to/de_results.csv")

✅ VERIFICATION: "✓ Data loaded successfully: N total genes, M DE genes (X up, Y down)"

Step 2 - Run integration analysis:

from run_integration_workflow import run_integration_workflow
results = run_integration_workflow(de_data, genome="hg38", output_dir="regulator_results")

DO NOT write inline API code or custom scoring. Just call the workflow function.

⏱️ This step takes 15-25 minutes (ChIP-Atlas API polling + target gene downloads).

✅ VERIFICATION: "✓ Integration analysis completed successfully!"

Step 3 - Generate visualizations:

from generate_all_plots import generate_all_plots
generate_all_plots(results, output_dir="regulator_results")

🚨 DO NOT write inline plotting code (ggplot, ggsave, etc.). Just use the script. 🚨

✅ VERIFICATION: "✓ All visualizations generated successfully!"

Step 4 - Export results:

from export_all import export_all
export_all(results, output_dir="regulator_results")

DO NOT write custom export code. Use export_all().

✅ VERIFICATION: "=== Export Complete ==="

⚠️ CRITICAL - DO NOT:

• ❌ Write inline API code → STOP: Use run_integration_workflow()
• ❌ Write inline plotting code → STOP: Use generate_all_plots()
• ❌ Write custom export code → STOP: Use export_all()
• ❌ Write custom Fisher's test code → STOP: Built into score_regulons()

⚠️ IF SCRIPTS FAIL - Script Failure Hierarchy:

1. Fix and Retry (90%) - Install missing package, re-run script
2. Modify Script (5%) - Edit the script file itself, document changes
3. Use as Reference (4%) - Read script, adapt approach, cite source
4. Write from Scratch (1%) - Only if genuinely impossible, explain why

NEVER skip directly to writing inline code without trying the script first.

Common Issues

Interpretation Guidelines

Regulatory Score

Combined evidence: -log10(Fisher P) × Concordance × -log10(ChIP Q)

Direction Classification

• Activator (concordance >60%, majority up): TF likely activates these genes
• Repressor (concordance >60%, majority down): TF likely represses these genes
• Mixed (concordance ≤60%): No clear directional bias — context-dependent regulation

Key Caveats

• Results biased toward well-studied TFs/cell types in ChIP-Atlas
• Binding enrichment ≠ regulatory causation (validate with perturbation)
• Directional labels assume simple activation/repression (ignores context-dependent regulation)
• Combined score is a heuristic ranking, not a formal multi-test correction
• Fisher's test assumes independence (may be violated if targets cluster in pathways)

Suggested Next Steps

After identifying upstream regulators:

• Validate binding: Use chip-atlas-target-genes to examine cell-type-specific binding patterns for top TFs
• Functional enrichment: Use functional-enrichment-from-degs on TF-target gene subsets
• Co-expression: Use gene-correlation-archs4 to check if TF and targets co-express
• Network inference: Use grn-pyscenic for single-cell GRN validation
• Literature review: Use literature-review to validate TF-disease associations

Related Skills

• chip-atlas-peak-enrichment - Component: TF binding enrichment analysis
• chip-atlas-target-genes - Component: TF target gene retrieval
• bulk-rnaseq-counts-to-de-deseq2 - Upstream: generates DE results input
• de-results-to-gene-lists - Upstream: generates filtered gene lists
• functional-enrichment-from-degs - Complementary: pathway-level enrichment

References

• Zou Z, et al. (2024) ChIP-Atlas 3.0: a gene regulation data-mining platform. Nucleic Acids Res. 52(W1):W159-W166
• Oki S, et al. (2018) ChIP-Atlas: a data-mining suite. EMBO Rep. 19(12):e46255
• Fisher RA (1922) On the interpretation of chi-squared. J R Stat Soc. 85(1):87-94