Chromatin*is*an*ancient*innovation*conserved*between*Archaea*and*Eukarya* *

It has been known since the 1990s that the DNA of archaea is wrapped around histones to form 10 complexes that closely resemble the nucleosomes found in eukaryotes, albeit with four rather 11 than eight histone subunits. Nucleosomes are the fundamental units of chromatin, the highly-12 ordered and compact structure that all the DNA in a cell is packed into. Now we know exactl

A comprehensive platform for highly multiplexed mammalian functional genetic screens

<p>Abstract</p> <p>Background</p> <p>Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens.</p> <p>Results</p> <p>Experiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA "dropout" screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens. Additional experiments demonstrated that GMAP is equivalent to similar microarray-based products when used for deconvolution of open reading frame over-expression screens.</p> <p>Conclusion</p> <p>Herein, we demonstrate four major applications for the GMAP resource, including deconvolution of pooled RNAi screens in cells with at least 90,000 distinct shRNAs. We also provide detailed methodologies for pooled shRNA screen readout using GMAP and compare next-generation sequencing to GMAP (i.e. microarray) based deconvolution methods.</p

Haloferax nucleosome centre positions

Archaeal browser track with 1-based nucleosome centre positions, bed fil

Haloferax volcanii reference genome

The Hfx. volcanii genome that we used to align read

Data from: Chromatin is an ancient innovation conserved between Archaea and Eukarya

The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ~147bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have long been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-free regions at transcriptional start sites and conserved 1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient

Nucleosome MNase-seq data aligned to Haloferax genome (BAM file)

Aligned nucleosome read

Nucleosome MNase-seq data aligned to Haloferax genome (BAI file)

Nucleosome MNase-seq data aligned to Haloferax genome (BAI file

Expression data for transcripts

FPKM and expression data for all transcript