ABSTRACT
CHD1 is a conserved chromatin remodeling factor that localizes to active genes and functions in nucleosome assembly and positioning as well as histone turnover. Mouse CHD1 is required for the maintenance of stem cell pluripotency while human CHD1 may function as a tumor suppressor. To investigate the action of CHD1 on higher order chromatin structure in differentiated cells, we examined the consequences of loss of CHD1 and over-expression of CHD1 on polytene chromosomes from salivary glands of third instar Drosophila melanogaster larvae. We observed that chromosome structure is sensitive to the amount of this remodeler. Loss of CHD1 resulted in alterations of chromosome structure and an increase in the heterochromatin protein HP1a, while over-expression of CHD1 disrupted higher order chromatin structure and caused a decrease in levels of HP1a. Over-expression of an ATPase inactive form of CHD1 did not result in severe chromosomal defects, suggesting that the ATPase activity is required for this in vivo phenotype. Interestingly, changes in CHD1 protein levels did not correlate with changes in the levels of the euchromatin mark H3K4me3 or elongating RNA Polymerase II. Thus, while CHD1 is localized to transcriptionally active regions of the genome, it can function to alter the levels of HP1a, perhaps through changes in methylation of H3K9.
Subject(s)
Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Histones/metabolism , Transcription Factors/metabolism , Animals , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Histones/genetics , Larva/genetics , Larva/metabolism , Transcription Factors/geneticsABSTRACT
The expression of many metazoan genes is regulated through controlled release of RNA polymerase II (Pol II) that has paused during early transcription elongation. Pausing is highly enriched at genes in stimulus-responsive pathways, where it has been proposed to poise downstream targets for rapid gene activation. However, whether this represents the major function of pausing in these pathways remains to be determined. To address this question, we analyzed pausing within several stimulus-responsive networks in Drosophila and discovered that paused Pol II is much more prevalent at genes encoding components and regulators of signal transduction cascades than at inducible downstream targets. Within immune-responsive pathways, we found that pausing maintains basal expression of critical network hubs, including the key NF-κB transcription factor that triggers gene activation. Accordingly, loss of pausing through knockdown of the pause-inducing factor NELF leads to broadly attenuated immune gene activation. Investigation of murine embryonic stem cells revealed that pausing is similarly widespread at genes encoding signaling components that regulate self-renewal, particularly within the MAPK/ERK pathway. We conclude that the role of pausing goes well beyond poising-inducible genes for activation and propose that the primary function of paused Pol II is to establish basal activity of signal-responsive networks.
Subject(s)
Drosophila melanogaster/growth & development , Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental , Gene Regulatory Networks , RNA Polymerase II/physiology , Transcriptional Activation , Animals , Drosophila melanogaster/immunology , Embryonic Stem Cells/metabolism , Immunity/genetics , Janus Kinases/metabolism , Mice , RNA Polymerase II/genetics , Signal Transduction , Transcription Factors/genetics , Transcription Factors/physiologyABSTRACT
Eukaryotic cytosine methylation represses transcription but also occurs in the bodies of active genes, and the extent of methylation biology conservation is unclear. We quantified DNA methylation in 17 eukaryotic genomes and found that gene body methylation is conserved between plants and animals, whereas selective methylation of transposons is not. We show that methylation of plant transposons in the CHG context extends to green algae and that exclusion of histone H2A.Z from methylated DNA is conserved between plants and animals, and we present evidence for RNA-directed DNA methylation of fungal genes. Our data demonstrate that extant DNA methylation systems are mosaics of conserved and derived features, and indicate that gene body methylation is an ancient property of eukaryotic genomes.
Subject(s)
Biological Evolution , DNA Methylation , Eukaryota/genetics , Genome, Fungal , Genome, Plant , Genome , Animals , Chlorophyta/genetics , CpG Islands , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Transposable Elements , Eukaryota/metabolism , Fungi/genetics , Fungi/metabolism , Gene Expression Regulation , Histones/metabolism , Phylogeny , Plants/genetics , Plants/metabolism , Promoter Regions, Genetic , Selection, Genetic , Transcription, GeneticABSTRACT
In this report we describe chd1 mutant alleles and show that the CHD1 chromatin-remodeling factor is important for wing development and fertility. While CHD1 colocalizes with elongating RNA polymerase II (Pol II) on polytene chromosomes, elongating Pol II can persist on chromatin in the absence of CHD1. These results clarify the roles of chromatin remodelers in transcription and provide novel insights into CHD1 function.
