Finish polishing

presentation.mkdn

@@ -237,6 +237,23 @@ Data generated by Sarah Lamere, published in GEO as
 
 ![](graphics/CD4-csaw/rulegraphs/rulegraph-all-RASTER100.png)
 
+## Questions to focus on
+
+::: incremental
+
+1. How do we define the "promoter region" for each gene? \vspace{10pt}
+2. How do these histone marks behave in promoter regions? \vspace{10pt}
+3. What can these histone marks tell us about T-cell activation and
+   differentiation?
+
+:::
+
+## First question
+
+\centering \LARGE
+
+How do we define the "promoter region" for each gene?
+
 ## Histone modifications occur on consecutive histones
 
 ![ChIP-seq coverage in IL2 gene[^lamerethesis]](graphics/presentation/LaMere-thesis-fig3.9-SVG-CROP.png){ height=65% }
@@ -271,7 +288,7 @@ Data generated by Sarah Lamere, published in GEO as
 
 ## Each histone mark has an "effective promoter radius"
 
-![Enrichment of peaks near promoters](graphics/CD4-csaw/Promoter-Peak-Distance-Profile-PAGE1-CROP.pdf)
+![Enrichment of peaks near promoters](graphics/presentation/Promoter-Peak-Distance-Profile-SVG.pdf)
 
 ## Peaks in promoters correlate with gene expression
 
@@ -285,25 +302,50 @@ Data generated by Sarah Lamere, published in GEO as
 \caption{Expression distributions of genes with and without promoter peaks}
 \end{figure}
 
-## The story so far
+## First question
+
+\centering \LARGE
+
+How do we define the "promoter region" for each gene?
+
+## Answer: Define the promoter region empirically!
 
 <!-- TODO: Left column: text; right column: flip through relevant image -->
 
-* H3K4me2, H3K4me3, and H3K27me3 occur on many consecutive histones in
-  broad regions across the genome
-* These enriched regions occur more commonly within a certain radius
-  of gene promoters
-* This "effective promoter radius" is consistent across all samples
-  for a given histone mark, but differs between histone marks
+:::::::::: {.columns}
+::: {.column width="50%"}
+
+* H3K4me2, H3K4me3, and H3K27me3 occur in broad regions across the
+  genome
+* Enriched regions occur more commonly near promoters
+* Each histone mark has its own "effective promoter radius"
 * Presence or absence of a peak within this radius is correlated with
   gene expression
-  
-. . .
 
-Next: Does the position of a histone modification within a gene
-promoter matter to that gene's expression, or is it merely the
-presence or absence anywhere within the promoter?
-  
+:::
+
+::: {.column width="50%"}
+\centering
+\only<1>{\includegraphics[width=\textwidth,height=0.7\textheight]{graphics/presentation/CCF-plots-A-SVG.png}}
+\only<2>{\includegraphics[width=\textwidth,height=0.7\textheight]{graphics/presentation/Promoter-Peak-Distance-Profile-SVG.pdf}}
+\only<3>{\includegraphics[width=\textwidth,height=0.7\textheight]{graphics/presentation/FPKM-by-Peak-Violin-Plots-A-SVG.png}}
+:::
+::::::::::
+
+## Next question
+
+\centering \LARGE
+
+How do these histone marks behave in promoter regions?
+
+::: notes
+
+Does the position of a histone modification within a gene promoter
+matter to that gene's expression, or is it merely the presence or
+absence anywhere within the promoter?
+
+:::
+
 ## H3K4me2 promoter neighborhood K-means clusters
 
 ![Cluster means for H3K4me2](graphics/presentation/H3K4me2-neighborhood-clusters-CROP.png){ height=70% }
@@ -385,7 +427,13 @@ presence or absence anywhere within the promoter?
 :::
 ::::::::::
 
-## Summary of promoter relative coverage findings
+## Current question
+
+\centering \LARGE
+
+How do these histone marks behave in promoter regions?
+
+## Answer: Presence and position both matter!
 
 ### H3K4me2 & H3K4me3
 
@@ -396,19 +444,25 @@ presence or absence anywhere within the promoter?
 
 ### H3K27me3
 
-* Depletion of H3K27me3 at TSS associated with elevated gene
+* Depletion of H3K27me3 at TSS $\Rightarrow$ elevated gene expression
+* Enrichment of H3K27me3 upstream of TSS $\Rightarrow$ *more* elevated
   expression
-* Enrichment of H3K27me3 upstream of TSS even more strongly associated
-  with elevated expression
-* Other coverage profiles not associated with elevated expression
+* Other coverage profiles: no association
+
+## Last question
+
+\centering \LARGE
+
+What can these histone marks tell us about T-cell activation and
+differentiation?
 
 ## Differential modification disappears by Day 14
 
-![Differential modification between naïve and memory samples at each time point](graphics/presentation/RCT-thesis-table2.4-A-SVG-CROP.png)
+![Differential modification between naïve and memory samples at each time point](graphics/presentation/RCT-thesis-table2.4-A-SVG-CROP.pdf)
 
 ## Differential modification disappears by Day 14
 
-![Differential modification between naïve and memory samples at each time point](graphics/presentation/RCT-thesis-table2.4-B-SVG-CROP.png)
+![Differential modification between naïve and memory samples at each time point](graphics/presentation/RCT-thesis-table2.4-B-SVG-CROP.pdf)
 
 ## Promoter H3K4me2 levels converge at Day 14
 
@@ -456,21 +510,19 @@ presence or absence anywhere within the promoter?
 :::
 ::::::::::
 
-<!-- TODO: Intro MOFA, motivate by showing uncorrected PCA -->
-
-## MOFA identifies cross-dataset patterns of variation
+## MOFA: cross-dataset factor analysis
 
-![MOFA factor analysis schematic[^mofa]](graphics/presentation/MOFA-fig1A-SVG.png)
+![MOFA factor analysis schematic[^mofa]](graphics/presentation/MOFA-fig1A-SVG.png){ height=70% }
 
 [^mofa]: [Argelaguet, Velten, et. al. (2018)](https://onlinelibrary.wiley.com/doi/abs/10.15252/msb.20178124)
 
-## MOFA LFs explain variation in all 4 data sets
+## Some factors are shared while others are not
 
 \centering
 
 ![Variance explained in each data set by each LF](graphics/presentation/MOFA-varExplained-matrix-A-CROP.png){ height=70% }
 
-## 3 LFs are shared across all 4 data sets
+## 3 factors are shared across all 4 data sets
 
 \centering
 
@@ -478,17 +530,25 @@ presence or absence anywhere within the promoter?
 
 ## MOFA LF5 captures convergence pattern
 
-![LF1 & LF4: time point effect; LF5: convergence](graphics/CD4-csaw/MOFA-LF-scatter-small.png){ height=70% }
+![LF1 & LF4: time point effect; LF5: convergence](graphics/presentation/MOFA-LF-scatter-wide.png)
 
-## What have we learned?
+<!-- { height=70% } -->
 
-* Almost no differential modification observed between naïve and
+## Last question
+
+\centering \LARGE
+
+What can these histone marks tell us about T-cell activation and
+differentiation?
+
+## Answer: Epigenetic convergence between naïve and memory!
+
+* Almost no differential histone modification observed between naïve and
   memory at Day 14, despite plenty of differential modification at
   earlier time points.
-* RNA-seq data and all 3 histone marks' ChIP-seq data all show
-  "convergence" between naïve and memory by Day 14 in the first 2 or 3
-  principal coordinates.
-* MOFA captures this convergence pattern in one of the latent factors,
+* Expression and 3 histone marks all show "convergence" between naïve
+  and memory by Day 14 in the first 2 or 3 principal coordinates.
+* MOFA captures this convergence pattern in a single latent factor,
   indicating that this is a shared pattern across all 4 data sets.
 
 <!-- ## Slide -->
@@ -496,34 +556,86 @@ presence or absence anywhere within the promoter?
 <!-- ![(Insert figure legend)](graphics/CD4-csaw/LaMere2016_fig8.pdf) -->
 
 
-## Takeaway 1: Each histone mark has an "effective promoter radius"
+## Questions to focus on
 
-* H3K4me2, H3K4me3, and H3K27me3 ChIP-seq reads are enriched in broad
-  regions across the genome, representing areas where the histone
-  modification is present
-* These enriched regions occur more commonly within a certain radius
-  of gene promoters
-* This "effective promoter radius" is specific to each histone mark
-* Presence or absence of a peak within this radius is correlated with
-  gene expression
+### How do we define the "promoter region" for each gene?
+
+Define empirically using peak-to-promoter distances; validate by
+correlation with expression.
+
+. . .
+
+### How do these histone marks behave in promoter regions?
+
+Location matters! Specific coverage patterns correlated with elevated
+expression.
+
+. . .
+
+### What can we learn about T-cell activation and differentiation?
+
+Epigenetic & expression state of naïve and memory converges late after
+activation, consistent with naïve differentiation into memory.
+
+## Further conclusions & future directions
+
+* "Effective promoter region" is a valid concept but "radius"
+  oversimplifies: seek a better definition
+
+* Coverage profiles were only examined in naïve day 0 samples: further
+  analysis could incorporate time and cell type
+
+* Coverage profile normalization induces degeneracy: adapt a better
+  normalization from peak callers like SICER
+
+* Unimodal distribution of promoter coverage profiles is unexpected
+
+## Further conclusions & future directions
+
+* Experiment was not designed to directly test the epigenetic
+  convergence hypothesis: future experiments could include cultured
+  but un-activated controls
+
+* High correlation between H3K4me3 and H3K4me2 is curious given they
+  are mutually exclusive: design experiments to determine the degree
+  of actual co-occurrence
   
-## Takeaway 2: Peak position within the promoter is important
-
-* H3K4me2 and H3K4me3 peaks are more strongly associated with elevated
-  gene expression the closer they are to the TSS, with a slight bias
-  toward downstream peaks.
-* H3K27me3 depletion at the TSS and enrichement upstream are both
-  associated with elevated expression, while other patterns are not.
-* In all histone marks, position of modification within promoter
-  appears to be an important factor in association with gene
-  expression
+## Implications for transplant biology
+
+::: incremental
+
+* Epigenetic regulation through histone methylation is surely involved
+  in immune memory
+
+* Can we stop memory cells from forming by perturbing histone
+  methylation?
+
+* Can we disrupt memory cell function during rejection by perturbing
+  histone methylation?
+
+* Can we suggest druggable targets for better immune suppression by
+  looking at epigenetically upregulated genes in memory cells?
+
+:::
+
+## Acknowledgements
+
+* My mentors, past and present: Drs. Terry Gaasterland, Daniel
+  Salomon, and Andrew Su
+
+* My committee: Drs. Nicholas Schork, Ali Torkamani, Michael
+  Petrascheck, and Luc Teyton.
+
+* My many collaborators in the Salomon Lab
+
+* The Scripps Genomics Core
+
+* My parents, John & Chris Thompson
+
+## {.plain}
+
+\centering
 
-## Takeaway 3: Expression & epigenetic state both converge at Day 14
+\huge
 
-* At Day 14, almost no differential modification observed between
-  naïve and memory cells
-* Naïve and memory converge visually in PCoA plots
-* Convergence is a shared pattern of variation across all 3 histone
-  marks and gene expression
-* This is consistent with the hypothesis that the naïve cells have
-  differentiated into a more memory-like phenotype by day 14.
+Questions?

slides-notes.org

@@ -23,21 +23,21 @@
   the trend in a simpler context
 
 ** TODO Content issues
-- [ ] Maybe show more preprocessing, batch correction, etc.
-- [ ] Tie conclusions back to intro, add future directions
+- [X] Maybe show more preprocessing, batch correction, etc.
+- [X] Tie conclusions back to intro, add future directions
 - [X] Intro slide for MOFA: how and why
 - [X] Too many bullet points. Don't need to write everything I say.
   Move to speaker notes.
 - [X] Intro the histone marks and why we picked them
 - [X] Brief 30 second intro to RNA-seq & ChIP-seq
 - [X] Rephrase 3 topics as questions/hypotheses
-- [ ] Talk about 3 main take-home points at the beginning as well as
+- [X] Talk about 3 main take-home points at the beginning as well as
   the end
+- [X] On summary slides, bullets on left, image on right for each
+  bullet
+- [X] Time was around 40 minutes, so add at least 5 minutes of content
 - [ ] Keep reserve slides at the end with cut figures in case they
   come in handy for answering audience questions
-- [ ] On summary slides, bullets on left, image on right for each
-  bullet
-- [ ] Time was around 40 minutes, so add at least 5 minutes of content
 
 * DONE Notes from Sabah