High-throughput functional testing of ENCODE segmentation predictions.

Area of Science:

• Genomics
• Epigenetics
• Molecular Biology

Background:

• Histone modification states are hypothesized to reflect genomic DNA regulatory activity.
• The ENCODE Project Consortium mapped histone modifications to segment the genome based on predicted regulatory functions.

Purpose of the Study:

• To experimentally measure the cis-regulatory activity of ENCODE's predicted genomic segments.
• To validate the predictive power of histone modification states for regulatory activity in specific cell types.

Main Methods:

• Assessed cis-regulatory activity of over 2000 ENCODE predictions in K562 leukemia cells.
• Tested genomic segments predicted as Enhancers, Weak Enhancers, Repressed elements, and H1-hESC-specific Enhancers.
• Analyzed histone modifications (H3K36me3, H3K27ac), DNase I hypersensitivity (HS), and transcription factor (TF) binding models.

Main Results:

• Both Enhancer and Weak Enhancer segments in K562 cells showed higher activity than negative controls.
• Surprisingly, Weak Enhancer segments exhibited higher activity than Enhancer segments.
• Lower levels of H3K36me3 and H3K27ac correlated with higher expression, partly explaining Weak Enhancer activity.
• DNase I hypersensitivity predicted active sequences, but TF binding models were necessary for identifying highly expressed sequences.
• Approximately 26% of ENCODE enhancer predictions demonstrated regulatory activity.

Conclusions:

• Histone modification states partially reflect cis-regulatory activity, but are not the sole determinants.
• Specific sequence preferences, particularly transcription factor binding sites, are the causal drivers of cis-regulatory activity.
• The study validates a significant portion of ENCODE's enhancer predictions while highlighting the importance of sequence-level features.