A chromatin code for alternative splicing involving a putative association between CTCF and HP1α proteins

Area of Science:

• Molecular Biology
• Epigenetics
• Genomics

Background:

• Alternative splicing is regulated by splicing factors and RNA polymerase II (RNAPII) elongation.
• Emerging evidence suggests chromatin, transcription, and protein factors form a regulatory network.
• CCCTC-binding factor (CTCF), Argonaute protein 1 (AGO1), and heterochromatin protein 1 (HP1) are implicated in chromatin-associated splicing and RNAPII elongation.

Purpose of the Study:

• To investigate whether CTCF, AGO1, and HP1 family members associate at the chromatin level to modulate alternative splicing.
• To identify the combinatorial code of protein and histone mark binding to chromatin associated with alternative splicing patterns.
• To develop a chromatin-based RNA map to explain splicing regulation.

Main Methods:

• Chromatin immunoprecipitation sequencing (ChIP-Seq) for CTCF, AGO1, HP1α, histone marks (H3K27me3, H3K9me2, H3K36me3), RNAPII, and 5-methylcytosine (5metC).
• Analysis of alternative splicing arrays from MCF7 and MCF10 cell lines.
• Machine Learning techniques to identify significant chromatin signal changes associated with splicing regulation.

Main Results:

• A chromatin-based RNA map was constructed, explaining 68.55% of regulated splicing events between MCF7 and MCF10 cell lines.
• The identified chromatin code involves HP1α, CTCF, AGO1, RNAPII, and histone marks around regulated exons, differentiating exon skipping and inclusion.
• Significant association of HP1α and CTCF activities was found near regulated exons, with a putative DNA binding site for HP1α.

Conclusions:

• A substantial number of alternative splicing events are regulated by chromatin, involving HP1α and CTCF association near regulated exons.
• Further evidence supports the involvement of HP1α and AGO1 in chromatin-related splicing regulation.