Epigenomics and Chromatin Accessibility Research

(c) How does a variant affect regulation? Use RegulomeDB_query_variant for a scored summary. Then build multi-layer evidence: UCSC_get_encode_cCREs (is the variant in a cCRE?), GTEx_get_single_tissue_eqtls (is it an eQTL?), jaspar_search_matrices (does it disrupt a TF motif?). No single layer is sufficient — see the variant reasoning section below.

(d) What genes are regulated by an element? Use GTEx_get_single_tissue_eqtls or GTEx_query_eqtl to find genes whose expression is associated with variants in the element. Use SCREEN_get_regulatory_elements with element_type="PLS"/"pELS"/"dELS" to classify element-to-promoter relationships.

Reasoning: Histone Marks

Use histone mark identity to guide tool queries and interpret results before fetching data.

• H3K4me3 = active promoter. If present without H3K27ac, promoter may be active but not hyperacetylated.
• H3K27ac = active enhancer or promoter. Strong signal = regulatory element is on.
• H3K4me1 = poised or active enhancer. Needs H3K27ac to confirm activity; H3K4me1 alone = poised.
• H3K27me3 = Polycomb repression. Gene is silenced by PRC2.
• H3K9me3 = constitutive heterochromatin. Region is structurally silenced.
• H3K36me3 = transcribed gene body. Confirms active elongation.

Bivalent promoter logic: If you observe H3K4me3 + H3K27me3 together at the same locus, the promoter is bivalent — poised but not active. This is common in stem cells and developmentally regulated genes. Do not report such genes as "actively transcribed." Use GTEx_get_expression_summary to check if the gene is actually expressed in the tissue of interest.

Inference rule: If a user asks about a mark you haven't queried yet, ask: does the mark you have found already answer the question? H3K4me3 in a region predicts active transcription; you may not need to also query H3K36me3 unless confirming elongation specifically.

Reasoning: eQTL Interpretation

An eQTL means variant X is statistically associated with expression of gene Y in tissue T. Before reporting eQTL results, apply this chain of reasoning:

1. Association ≠ causation. The variant may be in LD with the causal variant. Report effect size (NES) and p-value, not causality.
2. Check tissue specificity. Use GTEx_get_multi_tissue_eqtls to see whether the effect is shared across tissues (m-value near 1.0 in many tissues) or tissue-specific (m-value near 1.0 in only one tissue). Tissue-specific eQTLs are stronger candidates for cell-type-specific regulation.
3. Cross-reference with chromatin. Is the eQTL variant inside a cCRE? Use UCSC_get_encode_cCREs on the variant's coordinates. If yes, the variant likely acts through a regulatory element.
4. Check TF motif disruption. Use jaspar_search_matrices to find motifs overlapping the eQTL locus. If the variant alleles differ in motif score, it is a candidate causal variant.
5. Effect direction matters. Positive NES = reference allele increases expression. Negative NES = alternative allele decreases expression.

Reasoning: Variant Regulatory Impact

To assess a non-coding variant's regulatory impact, build evidence from multiple independent layers. No single layer is sufficient.

Layer 1 — RegulomeDB score: High probability (score 1a–2b) means convergent evidence from eQTL + TF binding + DNase. Score 4–7 means weak support. Use as a triage filter.

Layer 2 — Regulatory element overlap: Query UCSC_get_encode_cCREs at the variant's coordinates. If the variant falls in a cCRE (especially PLS or pELS), it is in a functional context.

Layer 3 — eQTL evidence: Query GTEx_get_single_tissue_eqtls for nearby genes. If the variant is a significant eQTL, the association supports regulatory function.

Layer 4 — TFBS disruption: Query jaspar_search_matrices for TFs with motifs at the locus. If the variant changes a high-information-content position in a motif, it is a strong functional candidate.

Synthesis rule: Report each layer separately. Convergence across 3+ layers = high-confidence regulatory variant. A single layer (e.g., eQTL alone) warrants caution.

Phase 0: Disambiguation

MyGene_query_genes: query (string). Converts gene symbols to Ensembl IDs and coordinates. Filter results by symbol == '<GENE>' — first hit may not match.

ensembl_lookup_gene: gene_id (Ensembl ID), species (REQUIRED, "homo_sapiens"). Returns chr/start/end.

Key format notes:

• GTEx requires versioned GENCODE IDs: ENSG00000012048.20
• RegulomeDB takes rsIDs: rs4994
• GTEx variant IDs: chr17_43705621_T_C_b38
• UCSC cCRE regions: chrom="chr17", start=7668421, end=7687490

Phase 1: Histone Modification & ChIP-seq

ENCODE_search_histone_experiments: target (histone mark), cell_type (or tissue alias), biosample_term_name (most explicit ENCODE ontology name), limit.

ENCODE anatomy term notes: "breast" → try "breast epithelium" or "mammary epithelial cell"; "brain" → "brain" works; if 0 results, append "tissue", "epithelium", or "cell".

result = tu.tools.ENCODE_search_histone_experiments(target="H3K27ac", cell_type="GM12878", limit=5)
# result["data"]["experiments"][0]["accession"] -> "ENCSR000AKC"

GEO_search_chipseq_datasets: Fallback for older or non-ENCODE ChIP-seq datasets.

Phase 2: Chromatin Accessibility & Architecture

ENCODE_search_chromatin_accessibility: cell_type, limit. Returns ATAC-seq experiments.

ENCODE_get_chromatin_state: cell_type, limit. Returns ChromHMM 15-state annotations (TssA, Enh, TssBiv, ReprPC, etc.). Use to confirm bivalent promoter state or enhancer classification.

ENCODE_search_rnaseq_experiments: assay_type (default "total RNA-seq"), biosample, limit. If 0 results, retry with assay_type="polyA plus RNA-seq".

GEO_search_rnaseq_datasets / GEO_search_atacseq_datasets: query, organism, limit (also max_results). GEO adds "ATAC-seq" automatically for the ATAC tool.

ReMap_get_transcription_factor_binding (CTCF): gene_name="CTCF", cell_type, limit. Returns ENCODE TF ChIP-seq experiments.

Phase 3: Regulatory Element Identification

SCREEN_get_regulatory_elements: gene_name, element_type (PLS/pELS/dELS/CTCF-only/DNase-H3K4me3), limit.

UCSC_get_encode_cCREs: chrom (REQUIRED), start (REQUIRED), end (REQUIRED), genome (default "hg38"). Returns cCREs with Z-scores for DNase, H3K4me3, H3K27ac, CTCF signals.

# cCREs near TP53
result = tu.tools.UCSC_get_encode_cCREs(chrom="chr17", start=7668421, end=7687490, genome="hg38")

ENCODE_search_annotations: annotation_type ("candidate Cis-Regulatory Elements" or "chromatin state"), biosample_term_name, organism, assembly, limit.

Phase 4: eQTL Analysis

GTEx_get_single_tissue_eqtls: gene_symbol. Returns all significant eQTLs across tissues with snpId, pValue, tissueSiteDetailId, nes (normalized effect size).

result = tu.tools.GTEx_get_single_tissue_eqtls(gene_symbol="BRCA1")
from collections import Counter
tissue_counts = Counter(e["tissueSiteDetailId"] for e in result["data"])

GTEx_query_eqtl: gene_symbol, tissue (tissueSiteDetailId), page (1-indexed), size. Use for a specific tissue.

GTEx_get_multi_tissue_eqtls: operation="get_multi_tissue_eqtls", gencode_id (versioned, REQUIRED). Returns per-variant m-values showing tissue-sharing. m-value near 1.0 = effect present; near 0.0 = absent.

result = tu.tools.GTEx_get_multi_tissue_eqtls(
    operation="get_multi_tissue_eqtls",
    gencode_id="ENSG00000012048.20"
)

GTEx_calculate_eqtl: operation="calculate_eqtl", gencode_id, variant_id (chr_pos_ref_alt_b38), tissue_site_detail_id. Works for non-significant pairs.

eQTL_list_datasets / eQTL_get_associations: EBI eQTL Catalogue. Use dataset_id (from list call), gene_id (Ensembl), variant. Complementary to GTEx.

Phase 5: Gene Expression Context

GTEx_get_expression_summary: gene_symbol. Recommended — auto-resolves GENCODE versions. Returns median TPM per tissue.

result = tu.tools.GTEx_get_expression_summary(gene_symbol="BRCA1")
top_tissues = sorted(result["data"], key=lambda x: x["median"], reverse=True)[:5]

GTEx_get_median_gene_expression: Requires operation="get_median_gene_expression" + exact versioned gencode_id. Use only when version precision is needed.

GTEx_get_tissue_sites: No params. Returns all tissueSiteDetailId values.

Phase 6: Transcription Factor Binding

jaspar_search_matrices: name (TF name), collection ("CORE"), tax_group ("vertebrates"), species ("9606"), page_size.

result = tu.tools.jaspar_search_matrices(name="CTCF", collection="CORE", page_size=5)

jaspar_get_matrix: Returns position frequency matrix for a JASPAR matrix ID. Use to check if a variant allele disrupts a high-information-content position.

ReMap_get_transcription_factor_binding: gene_name (TF), cell_type, limit. Same tool used for CTCF in Phase 2 — applies to any TF.

STRING_get_functional_annotations: identifiers (gene name), species (9606), category ("Process"/"Function"/"KEGG"). Returns GO/KEGG/Reactome annotations for regulatory context.

Phase 7: Variant Regulatory Scoring

RegulomeDB_query_variant: rsid (e.g., "rs4994"). Returns probability, ranking (1a = strongest, 7 = weakest), and tissue-specific scores.

result = tu.tools.RegulomeDB_query_variant(rsid="rs4994")
score = result["data"]["regulome_score"]
# score["ranking"]: "1a" (eQTL + TF + motif + DNase) ... "7" (no evidence)
# score["probability"]: 0.0–1.0
top_tissues = sorted(score["tissue_specific_scores"].items(), key=lambda x: float(x[1]), reverse=True)[:5]

Rankings 1a–1f all have eQTL evidence. Rankings 2a–3b have TF binding without eQTL. Rankings 4–7 have decreasing evidence. Use ranking <= 2b as a threshold for "strong regulatory support."

Phase 8: Integration

Combine evidence tiers before reporting:

• T1 (Direct experimental): ENCODE ChIP-seq experiments, GTEx eQTL p < 5e-8
• T2 (Strong computational): RegulomeDB score <= 2, SCREEN cCRE classification, ChromHMM state
• T3 (Moderate): eQTL p < 0.05, JASPAR motif match, multi-tissue m-value > 0.5
• T4 (Annotation-based): STRING GO terms, literature references

Convergence of T1+T2 evidence from independent sources (e.g., ENCODE ChIP-seq overlapping a RegulomeDB 1a variant with GTEx eQTL) constitutes strong evidence for regulatory function. Contradictions between layers (e.g., high RegulomeDB score but no eQTL) should be explicitly noted.

Fallback Strategies