Question 4: Contrast of Global vs. Site-Specific Epigenetic Analysis Dr. Yan Yan's research on nanoscale shape utilized both bottom-up mass spectrometry and ChIP-seq to investigate histone modifications. Task: Contrast the information provided by these two tools. Explain why mass spectrometry was sufficient to identify the global decrease of H3K27me3 in response to spiky nanoparticles (GNP2), but why ChIP-seq was necessary to link these changes to the "mechanical sensing" of the cell and the reprogramming of specific synapse-related gene pathways.

What two tools did Dr. Yan Yan use to investigate histone modifications in response to nanoparticle shape?

Bottom-up mass spectrometry and ChIP-seq.

What does bottom-up mass spectrometry measure?

The relative abundance of modified histone peptides across all histones (H2A, H2B, H3, H4).

What is the main output of bottom-up mass spectrometry?

It identifies which histone modifications (methylation, acetylation) are changing and by how much globally.

What is the main limitation of bottom-up mass spectrometry?

It does not tell you where in the genome the modifications are located.

What does ChIP-seq provide that mass spectrometry cannot?

Site-specific resolution, mapping exactly which genomic regions and genes are associated with a specific histone mark.

How does ChIP-seq work?

Antibodies pull down DNA fragments associated with a specific mark (e.g., H3K27me3), then those fragments are sequenced and mapped back to the genome.

Why was mass spectrometry sufficient to identify the global decrease of H3K27me3 in response to spiky nanoparticles (GNP2)?

Because mass spectrometry measures the overall abundance of H3K27me3 across the whole sample, and it showed a significant reduction specifically with GNP2 compared to spherical GNP1.

What did the global decrease of H3K27me3 tell the researchers?

That the spiky nanoparticle shape was inducing a broad, systemic epigenetic response.

Why was ChIP-seq necessary to link H3K27me3 changes to mechanical sensing and synapse-related pathways?

Because ChIP-seq identified the physical location of the modifications, allowing gene annotation and pathway analysis.

What is "peak calling" in ChIP-seq analysis?

Identifying specific genomic regions where a histone mark is differentially present between conditions.

What did GO-term enrichment analysis reveal about the genes losing H3K27me3 in response to spiky nanoparticles?

That they were strongly associated with trans-synaptic signalling, sensory perception, and mechanical stimuli.

What hypothesis did the ChIP-seq mapping support?

That the cell "sees" the spiky nanoparticle shape through a mechanical sensing interface (a "bionanosynapse").

What would the researchers have missed if they only used mass spectrometry without ChIP-seq?

They would have known H3K27me3 was decreasing globally but would not have discovered that the cell was specifically reprogramming sensory and synaptic gene networks in response to physical stress from the spik