Canella 2012, ChIP-seq analysis of histone modifications, PolII and PolIII in liver

Description

The genomic loci occupied by RNA polymerase (pol) III have been characterized in human culture cells by genome-wide chromatin immunoprecipitation experiments followed by deep sequencing (ChIP-Seq). These studies have in particular shown that only about 40 % of the annotated 622 human tRNA genes and pseudogenes are occupied by pol III, and that these genes are often in regions of open chromatin rich in active pol II transcription units. Here we have used ChIP-Seq to characterize pol III-occupied loci in a differentiated tissue, the mouse liver. Our studies define the mouse liver pol III-occupied loci and point to a conserved pol III-occupied mammalian interspersed repeat (MIR) as a potential regulator of a pol III subunit-encoding gene. They reveal that synteny relationships can be established between a number of human and mouse pol III genes, and that the expression levels of these genes are significantly linked. They establish that variations within the A and B promoter boxes, as well as the strength of the terminator sequence can strongly affect pol III occupancy of tRNA genes. They reveal correlations with various genomic features that together describe the pol III occupancy scores over some 50% of tRNA genes. In mouse liver, pol III-occupied loci represented in the NCBI37/mm9 genome assembly comprise fifty 5S genes, fourteen known non-tRNA genes, nine 4.5S genes, and some twenty nine SINEs. In addition, out of the 433 annotated tRNA genes, half are occupied by pol III. Transfer RNA gene expression levels reflect both an underlying genomic organization that is conserved in dividing human culture cells and resting mouse liver cells, and the particular promoter and terminator strengths of individual genes.

Overall design

12 samples examinded, 4 on pol III, 2 on pol II, 2 on H3K4me3, 2 on H3k36me3, 2 input samples.

Source

Samples

Technical Notes:

SRA files were downloaded from GEO and processed using the following bash commands:

1. Extract FASTQ from SRA file:
   

   fastq-dump SAMPLE.sra
   

2. Map reads to genome using Bowtie:
   

   bowtie --best --strata -m1 --sam -l 36 -n 3 h_sapiens_ncbi36 -q SAMPLE.fastq > SAMPLE.sam
   

3. Clean the results from unmapped reads:
   

   awk 'BEGIN {FS="\t"} $3 != "\*" {print $0}' SAMPLE.sam > SAMPLE_clean.sam
   

4. Make BAM file:
   

   samtools view -bS -o SAMPLE.bam SAMPLE_clean.sam
   

5. Sort it:
   

   samtools sort SAMPLE.bam SAMPLE_sorted
   

6. Make BED file:
   

   bamToBed -i SAMPLE_sorted.bam > SAMPLE.bed
   

7. Make SGA file:
   

   bed2sga.pl -s hg18 -f FEATURE < SAMPLE.bed | sort -s -k1,1 -k3,3n -k4,4 | compactsga > SAMPLE.sga
   

References

1. Canella D, Bernasconi D, Gilardi F, LeMartelot G, Migliavacca E, Praz V et al.
   A multiplicity of factors contributes to selective RNA polymerase III occupancy of a subset of RNA polymerase III genes in mouse liver. Genome Res. 2012 Apr;22(4):666-80.
   PMID: 22287103

2. GEO series GSE33421 A Multiplicity of Factors Contributes to Selective RNA polymerase III occupancy of a Subset of RNA polymerase III Genes in Mouse Liver.