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Elevated temperatures activate a heat shock response (HSR) to protect cells from the pathological effects of protein mis-folding, cellular mis-organization, organelle dysfunction and altered membrane fluidity. This response includes activation of the conserved transcription factor heat shock factor 1 (HSF-1), which binds heat shock elements (HSEs) in the promoters of genes induced by heat shock (HS). The upregulation of protein-coding genes (PCGs), such as heat shock proteins and cytoskeletal regulators, is critical for cellular survival during elevated temperatures. While the transcriptional response of PCGs to HS has been comprehensively analyzed in a variety of organisms, the effect of this stress on the expression of non-coding RNAs (ncRNAs) has not been systematically examined. Here we show that in Caenorhabditis elegans HS induces up- and downregulation of specific ncRNAs from multiple classes, including miRNA, piRNA, lincRNA, pseudogene and repeat elements. Moreover, some ncRNA genes appear to be direct targets of the HSR, as they contain HSF-1 bound HSEs in their promoters and their expression is regulated by this factor during HS. These results demonstrate that multiple ncRNA genes respond to HS, some as direct HSF-1 targets, providing new candidates that may contribute to organismal survival during this stress.

Heat Shock Factor 1 (HSF-1) is a key regulator of the heat shock response (HSR). Upon heat shock, HSF-1 binds well-conserved motifs, called Heat Shock Elements (HSEs), and drives expression of genes important for cellular protection during this stress. Remarkably, we found that substantial numbers of HSEs in multiple Caenorhabditis species reside within Helitrons, a type of DNA transposon. Consistent with Helitron-embedded HSEs being functional, upon heat shock they display increased HSF-1 and RNA polymerase II occupancy and up-regulation of nearby genes in C. elegans. Interestingly, we found that different genes appear to be incorporated into the HSR by species-specific Helitron insertions in C. elegans and C. briggsae and by strain-specific insertions among different wild isolates of C. elegans. Our studies uncover previously unidentified targets of HSF-1 and show that Helitron insertions are responsible for rewiring and diversifying the Caenorhabditis HSR.

Elevated temperatures activate a Heat Shock Response (HSR) to protect cells from the pathological effects of protein mis-folding, cellular mis-organization, organelle dysfunction and altered membrane fluidity. This response includes activation of the conserved transcription factor Heat Shock Factor 1 (HSF-1), which binds Heat Shock Elements (HSEs) in the promoters of genes induced by heat shock (HS). The up-regulation of protein-coding genes (PCGs), such as Heat Shock Proteins (HSPs) and cytoskeletal regulators, is critical for cellular survival during elevated temperatures. While the transcriptional response of PCGs to heat shock has been comprehensively analyzed in a variety of organisms, the effect of this stress on the expression of non-coding RNAs (ncRNAs) has not been systematically examined. Here we show that inCaenorhabditis elegansHS induces up- and down-regulation of specific ncRNAs from multiple classes, including miRNA, piRNA, lincRNA, pseudogene, and repeat elements. Moreover, some ncRNA genes appear to be direct targets of the HSR, as they contain HSF-1 bound HSEs in their promoters and their expression is regulated by this factor during HS. These results demonstrate that multiple ncRNA genes respond to HS, some as direct HSF-1 targets, providing new candidates that may contribute to organismal survival during this stress.

Formation of heterochromatin serves a critical role in organizing the genome and regulating gene expression. In most organisms, heterochromatin flanks centromeres and telomeres. To identify heterochromatic regions in the heavily studied model C. elegans, which possesses holocentric chromosomes with dispersed centromeres, we analyzed the genome-wide distribution of the heterochromatin protein 1 (HP1) ortholog HPL-2 and compared its distribution to other features commonly associated with heterochromatin. HPL-2 binding highly correlates with histone H3 mono- and dimethylated at lysine 9 (H3K9me1 and H3K9me2) and forms broad domains on autosomal arms. Although HPL-2, like other HP1 orthologs, binds H3K9me peptides in vitro, the distribution of HPL-2 in vivo appears relatively normal in mutant embryos that lack H3K9me, demonstrating that the chromosomal distribution of HPL-2 can be achieved in an H3K9me-independent manner. Consistent with HPL-2 serving roles independent of H3K9me, hpl-2 mutant worms display more severe defects than mutant worms lacking H3K9me. HPL-2 binding is enriched for repetitive sequences, and on chromosome arms is anticorrelated with centromeres. At the genic level, HPL-2 preferentially associates with well-expressed genes, and loss of HPL-2 results in up-regulation of some binding targets and down-regulation of others. Our work defines heterochromatin in an important model organism and uncovers both shared and distinctive properties of heterochromatin relative to other systems.

The DREAM (Dp/Retinoblastoma(Rb)-like/E2F/MuvB) transcriptional repressor complex acts as a gatekeeper of the mammalian cell cycle by establishing and maintaining cellular quiescence. How DREAM’s three functional components, the E2F-DP heterodimer, the Rb-like pocket protein, and the MuvB subcomplex, form and function at target gene promoters remains unknown. The current model invokes that the pocket protein links E2F-DP and MuvB and is essential for gene repression. We tested this model by assessing how the conserved yet less redundant DREAM system in Caenorhabditis elegans is affected by absence of the sole C. elegans pocket protein LIN-35. Using a LIN-35 protein null mutant, we analyzed the assembly of E2F-DP and MuvB at promoters that are bound by DREAM and the level of expression of those "DREAM target genes" in embryos. We report that LIN-35 indeed mediates the association of E2F-DP and MuvB, a function that stabilizes DREAM subunit occupancy at target genes. In the absence of LIN-35, the occupancy of E2F-DP and MuvB at most DREAM target genes decreases dramatically and many of those genes become upregulated. The retention of E2F-DP and MuvB at some target gene promoters in lin-35 null embryos allowed us to test their contribution to DREAM target gene repression. Depletion of MuvB, but not E2F-DP, in the sensitized lin-35 null background caused further upregulation of DREAM target genes. We conclude that the pocket protein functions primarily to support MuvB-mediated repression of DREAM targets and that transcriptional repression is the innate function of the evolutionarily conserved MuvB complex. Our findings provide important insights into how mammalian DREAM assembly and disassembly may regulate gene expression and the cell cycle., Author summary The 8-subunit DREAM transcriptional repressor complex contains 3 functional components that together control expression of cell cycle and developmental genes. How the E2F-DP transcription factor heterodimer, the pocket protein, and the highly conserved MuvB complex coalesce on chromatin and repress DREAM target genes has yet to be determined. We directly tested the prevailing model that the DREAM pocket protein links E2F-DP to MuvB and is required for gene repression. Using a protein null mutant of the sole C. elegans pocket protein LIN-35, we demonstrate that the pocket protein indeed links E2F-DP and MuvB, which aids in the stable occupancy of DREAM components near target genes. Depletion of additional DREAM components in lin-35 null worms revealed that the remaining chromatin-bound MuvB represses target genes. We conclude that the MuvB subcomplex mediates DREAM’s critical repressive function. Our functional genomics approach in the simplified C. elegans system reveals that the ancestral function of the pocket protein is to stabilize the innate repressive activity of MuvB, ensuring proper regulation of DREAM target genes through development.

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal ‘arms’, and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function., National Science Foundation (U.S.) (1122374)