Question of heatmap of ChIP-seq

Question

[lida]

Hello ,

I'm dealing with a ChIP-seq data of H3K4me3 modification from drosophila and make a heatmap by deeptools-3.3.0 with following commonds

First, I extract the chromosome number, chrStart, chrEnd, 6, strand and 8 columns from annotated information to make a BED file for computeMatrix

cat dmel-all-r6.19.gtf |awk 'BEGIN{OFS="\t"} {if ($3=="gene") print "chr"$1,$4,$5,$6,$7,$8}'>dm_genes.gtf

Then I use --referencePoint TSS mode and +/-2k as promoter region for computeMatrix and plot the heatmap

computeMatrix reference-point -S 3-11.scale.bw -R dm_genes.gtf -o signal_heatmap/3-11_gtf.gz --referencePoint TSS -a 2000 -b 2000 --skipZeros --missingDataAsZero -p 16

plotHeatmap -m signal_heatmap/3-11_gtf.gz -out signal_heatmap/3-11_gtf.png --colorList white,steelblue --sortUsing max

The resulting plot is as follows:

There is a gap in TSS, which I consider is not that common for H3K4me3 modification at TSS. I'm quite new for ChIP-seq analysis as well as epigentic study of histone modification, so I want to know whether I create the heatmap properly, specificly for how I treat the promoter region.

Any help would be appreciated, Thanks!

Answer 1

[ATpoint]

The nucleosome-free at the TSS is deprived of histones and therefore you don't get a ChIP signal there for histone modifications.

See from https://www.sciencedirect.com/science/article/pii/S0968000410000940 this image:

[lida]

Thanks for your reply.

But the situation you mentioned above is describing a majority of genes or just the active genes? As your figure demonstrated, there is some histone modifications which cover the TSS for inactive gene.

By the way, is it common for using the chromosome Start coordinate of annotated genes and its upstream/downstream region to set the promoter window?

[ATpoint]

By the way, is it common for using the chromosome Start coordinate of annotated genes and its upstream/downstream region to set the promoter window?

Afaik yes, but mind that genes on the minus strand would reqiure the end coordinate to define promoters. Actually TSS is better than gene coordinates to capture alternative promoters of isoforms.

[lida]

Again, Thanks for your help, I will try your suggestion :)