Egelhofer, Thea et al. (2011) International Worm Meeting "An assessment of the quality of histone-modification antibodies."

Strome, Susan, Perry Lab, Mark, Pirrotta Lab, Vincenzo, Lieb, Jason, Lee, Kyungjoon, Kolasinska-Zwierz, Paulina, Karpen Lab, Gary, Park Lab, Peter, Minoda, Aki, Klugman, Sarit, Kuroda Lab, Mitzi, Elgin Lab, Sarah, Ren Lab, Bing, Hawkins, R. David, Ahringer Lab, Julie, Egelhofer, Thea

[International Worm Meeting, 2011]

Investigations of transcriptional regulation and chromatin organization often rely heavily on antibodies to various post-translational modifications on histones. The reproducibility and biological relevance of histone-modification studies depends on the specificity and performance of the antibodies, most of which are now provided commercially. As part of our activities in the NIH modENCODE and Roadmap Reference Epigenome initiative, we are using antibodies in conjunction with chromatin immunoprecipitation (ChIP) to determine the genomic distributions of histone modifications in C. elegans, Drosophila, and human cells. We have tested the specificity and utility of 246 antibodies raised against 3 unmodified histones and 57 different histone modifications (Egelhofer et al. Nature Struc & Molec Biol 18: 91-93). Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot analysis. Among specific antibodies, more than 20% failed in ChIP experiments. We discuss our tests of antibody specificity and our criteria for successful performance. We advise rigorous testing of histone-modification antibodies in the organism of interest before use, and we provide a website for posting new results (http://compbio.med.harvard.edu/antibodies/).

Garrigues, Jacob et al. (2011) International Worm Meeting "Investigating the roles of a worm HP1 homolog during embryogenesis."

Strome, Susan, Egelhofer, Thea, Garrigues, Jacob

[International Worm Meeting, 2011]

Proper formation of the germ line during animal development is essential for the propagation of species. In C. elegans, a gene that encodes a homolog of Heterochromatin Protein 1 (HP1), hpl-2, is required for a functional germ line at restrictive temperature (1). While previously reported that a null allele of hpl-2 has a maternal-effect sterile (Mes) phenotype (2), we found that this sterility can be rescued by zygotic expression of hpl-2(+), suggesting that HPL-2 serves a critical role during embryogenesis. The observed Mes phenotype and the possibility, suggested by findings in Arabidopsis (3), that HPL-2 may bind histone H3 trimethylated at lysine 27 (H3K27me3) generated by the Polycomb-like repressive complex MES-2/3/6 prompted us to assess the distribution of HPL-2 genome-wide in embryos, by chromatin immunoprecipitation followed by microarray analysis (ChIP-chip). The distribution of HPL-2 does not correlate with H3K27me3, but instead correlates well with H3K9me2, is highly enriched on autosome "arms", and is depleted from the X chromosome. At the gene level, HPL-2 binds genes with developmental roles, suggesting a direct regulatory role in their expression; many of these genes are associated with reproduction. Interestingly, a subset of HPL-2 peaks overlap with peaks of SET-2 (also obtained by ChIP-chip), a histone methyltransferase that generates much of the H3K4me signal associated with transcription initiation in worms. Furthermore, loss of HPL-2 results in increased H3K4me3 levels at these overlapping regions, suggesting that one role of HPL-2 is to antagonize the activity of SET-2 at transcription start sites. This may explain the genetic interaction between hpl-2 and set-2 previously observed (4), and may demonstrate a new role for HP1 proteins during development. 1. Couteau et al. (2002) EMBO Rep 3, 235-241. 2. Coustham et al. (2006) Dev Biol 297, 308-322. 3. Zhang et al. (2007) Nat Struct Mol Biol 14, 869-871. 4. Simonet et al. (2007) Dev Biol 312, 367-383.

Larson, Braden John et al. (2021) International Worm Meeting "Redundant Mechanisms of X Chromosome Repression in the C. elegans Male Germline"

Larson, Braden John, Strome, Susan, Egelhofer, Thea

[International Worm Meeting, 2021]

Organization of the genome into domains of euchromatin and heterochromatin is a conserved feature of all eukaryotes and precise regulation of these domains is important for organism health and development. Proper formation of heterochromatin is crucial for transcriptional repression, chromosome segregation, and maintenance of genome integrity. Heterochromatin can be categorized as either facultative or constitutive. These two types of heterochromatin are often distinguished by their associated histone modifications: methylation of lysine 27 or lysine 9 on histone H3. H3K27me is associated with facultative heterochromatin, and its domains are found throughout genomes, often with developmentally regulated genes. H3K9me is associated with constitutive heterochromatin, and is generally concentrated in gene-poor, repeat-rich regions such as pericentric regions. Thus, anticorrelation of H3K27me and H3K9me domains is observed in many model organisms. However, in C. elegans, H3K27me and H3K9me domains show a surprising amount of positive correlation, suggesting a species-specific mechanism for organizing facultative and constitutive heterochromatin. In the C. elegans germline, H3K27me is enriched on the X chromosome, and its loss leads to sterility in the F2 generation. Interestingly, H3K27me(-) F2 males that inherit a paternal X chromosome (Xp) and no maternal X chromosome (Xm) are usually fertile. The fertility of these males is dependent on H3K9me, which is enriched on the single X chromosome in the male germline. These observations suggest a potential redundant function of H3K27me and H3K9me in repressing the X chromosome in the male germline to promote fertility in subsequent generations. We are investigating the organization and functions of H3K27me and H3K9me in the C. elegans male germline. By generating chimeric animals whose germlines inherit only paternal chromosomes (XpXp), we've shown that a double dose of paternal chromosomes lacking H3K27me can also support fertile hermaphrodite germline development in an H3K9me-dependent manner. We are comparing misexpression of genes and repetitive elements in hermaphrodite and male germlines lacking H3K27me, H3K9me, or both. Lastly, we are using CUT&RUN to examine the distribution of H3K27me in germ cells and to test whether H3K9me regulates this distribution, as it does in Mouse and Neurospora.

Takasaki, Teruaki et al. (2013) International Worm Meeting "MRG-1 acts as an epigenome interpreter of Lys36 methylation on histone H3."

Sakamoto, Hiroshi, Strome, Susan, Takasaki, Teruaki, Rechtsteiner, Andreas, Egelhofer, Thea

[International Worm Meeting, 2013]

We previously identified the autosome-associated protein MRG-1 as an essential maternal factor for proper germline development, yet its molecular function remains unclear (Takasaki et al., 2007). MRG-1 contains a CHROMO domain, which is found in numerous proteins that associate with methylated histones. In our search for the element(s) that recruits MRG-1 to autosomes, we found that oocyte and sperm chromosomes arrive in the zygote marked with methylated H3K36, and that MRG-1 accumulates predominantly on those methylated chromosomes. Genome-wide chromatin immunoprecipitation analysis in early embryos revealed that methylation of H3K36 is propagated on the body of germline-expressed genes by the histone methyltransferase MES-4 in an epigenetic manner (Rechtsteiner et al., 2010; Furuhashi et al., 2010). The distribution of MRG-1 exhibits a high correlation with the distributions of both MES-4 and H3K36me, and the accumulation of MRG-1 on germline-specific genes depends on MES-4-generated H3K36me. These findings suggest that MRG-1 recognizes germline-expressed genes using the epigenetic mark H3K36me generated by MES-4. Strikingly, we found that germline-specific loci exhibit an atypical H4K16 acetylation pattern, which is dependent on MES-4. By analogy to the Drosophila homolog of MRG-1, which acts in the dosage compensation complex (the MSL complex) to generate H4K16ac on the X chromosome, it is likely that MRG-1 contributes to the germline-gene-specific H4K16ac pattern. Supporting this idea, we identified MYS-2, which is a homolog of another component of the MSL complex, as an H4K16 acetyltransferase. Furthermore, we found that loss of MYS-2, like loss of MES-4 and MRG-1, suppresses ectopic expression of germline genes in somatic cells of mep-1 mutant larvae. This suggests that H4K16 acetylation serves a pivotal role in germline development. Taken together, our results suggest that MRG-1 interprets the epigenetic H3K36me landmark and as part of an MSL-like complex generates H4K16ac specifically at germline-expressed genes.

Knutson, Andrew et al. (2015) International Worm Meeting "P granules maintain germ cell identity in the C. elegans germline."

Strome, Susan, Egelhofer, Thea, Rechtsteiner, Andreas, Knutson, Andrew, Tuckfield, Lynnia

[International Worm Meeting, 2015]

Like stem cells, germ cells are able to self renew and, after the union of 2 gametes, differentiate into a multitude of cell types. The factors and mechanisms employed by germ cells to maintain their broad differentiation potential are only starting to be understood and involve regulation at both the transcriptional and translational levels. In C. elegans germ cells, cytoplasmic ribonucleoprotein structures termed P granules participate in maintaining germ cell totipotency (Updike et al. Current Biology 24: 970-975, 2014). Removal of 4 major P-granule proteins (PGL-1, PGL-3, GLH-1, and GLH-4) via RNAi causes sterility and underproliferated germlines. Remarkably, a fraction of these germlines also express neuronal markers (unc-119::GFP and unc-33::GFP) and a muscle marker (MYO-3). This suggests that P granules prevent somatic development in the germline (Updike et al. 2014). We have found that glh-1 single mutants and pgl-1 single RNAi germlines express the unc-119::gfp transgene and stain for MYO-3, revealing the importance of single P-granule proteins in maintaining germ cell totipotency. Additionally, we see a loss of germline characteristics upon P-granule depletion, including loss of the mitotic factor REC-8 and the meiotic factor HTP-3. We also see cellularization in the normally syncytial region of the germline. Taken together, these findings suggests that P-granule-compromised germ cells are differentiating away from a germ cell fate and toward somatic fates. To better understand the spectrum of somatic genes that are expressed in P-granule-compromised germlines and the extent of somatic development, we are performing RNA-seq analysis of dissected germlines that have been depleted of P granules by RNAi and that express the unc-119::gfp neuronal reporter. This analysis may reveal important master regulator transcripts that must be sequestered/silenced/degraded by P granules to prevent premature somatic development. We hypothesize that P granules prevent the translation of somatic transcripts that are stochastically expressed in the germline. P granules may intercept and/or process these mRNAs as they exit the nucleus, thus maintaining germ cell fate and totipotency. .

Costello, Meghan Elizabeth et al. (2013) International Worm Meeting "synMuv B regulation of chromatin states at high temperature."

Egelhofer, Thea, Petrella, Lisa N, Rechtsteiner, Andreas, Strome, Susan, Costello, Meghan Elizabeth

[International Worm Meeting, 2013]

Most organisms experience variations in temperature throughout their lifespan and thus require mechanisms to buffer gene expression in response to fluctuations in temperature. It has been observed that during development, chromatin states are influenced by temperature. For example, in Drosophila heterochromatin formation and spreading is partially compromised at high temperatures. In order to understand the effect of temperature during development, it is critical to understand how chromatin structure affects the buffering of gene expression at elevated temperatures. We are investigating changes in chromatin state at high temperature through study of the C. elegans synMuv B chromatin regulators. The synMuv B proteins include Retinoblastoma/LIN-35 and other members of the DRM complex and HPL-2, the worm homolog of HP1. Mutations in many synMuv B genes cause high temperature larval arrest (HTA) due to derepression of germline gene expression in somatic cells, most notably the intestine. Loss of germline chromatin modifiers suppresses somatic expression of germline genes in synMuv B mutants and rescues the HTA phenotype. This suggests that changes in chromatin states in the soma underlie the temperature-sensitive gene misexpression in synMuv B mutants. Consistent with this hypothesis, immunostaining of synMuv B mutants revealed altered levels of specific histone modifications in somatic tissues. To investigate changes in active and repressive chromatin marks at high resolution genome-wide in synMuv B mutants, we have performed ChIP-seq experiments in lin-35, lin-37, and lin-15B mutants at both low and high temperature. Additionally, in order to determine if the absence of wild-type synMuv B proteins causes the loss of a closed chromatin state at elevated temperatures, we are using the lacO/LacI system to visualize changes in chromatin compaction in lin-15B and lin-35 mutants.

Fealey, Meghan E et al. (2017) International Worm Meeting "Developmental regulation of H3K9me2 and chromatin compaction by C. elegans synMuv B proteins."

Petrella, Lisa N, Strome, Susan, Fealey, Meghan E, Egelhofer, Thea, Rechsteiner, Andreas, Doerger, Ethan

[International Worm Meeting, 2017]

A central question in development is how chromatin is organized and regulated to ensure proper gene expression and cell fate. During early embryo development, before gene expression is globally upregulated, chromatin is found in an open state. As development proceeds and cells differentiate, the genome compacts and becomes organized into open and closed domains. Genes required for a particular fate remain open and poised for transcription factor binding, while genes not needed for that fate are further compacted and sequestered to the nuclear periphery. Although this process is highly regulated, many of the proteins involved in this progression are unknown. Loss of C. elegans synMuv B proteins causes changes in the developmental regulation of chromatin and gene expression. Many synMuv B mutants, including lin-15B, show ectopic expression of germline genes in somatic cells and a high temperature larval arrest (HTA) phenotype. The HTA phenotype is rescued by knockdown of chromatin modifiers, suggesting that synMuv B proteins regulate gene expression programs epigenetically. To investigate ways in which synMuv B proteins may affect chromatin, we performed ChIP-seq analysis of several histone modifications in wild type and synMuv B mutants at 20 deg C and 26 deg C. Intriguingly, lin-15B mutants lose promoter localized H3K9me2 over germline genes in somatic tissues in a temperature independent manner. Typical enrichment of H3K9me2 over gene bodies is not disrupted in these mutants. Promoter localized H3K9me2 is an unstudied pattern for this mark in C. elegans, and the mechanism by which it regulates gene expression is unknown. We investigated if synMuv B proteins function to regulate developmental chromatin compaction. synMuv B mutants display a developmental delay in chromatin compaction that is sensitive to temperature. This delay results in open chromatin during the window when ectopic expression of germline genes in somatic tissues begins. Using temperature shift assays, we found that the crucial developmental time period for the HTA phenotype is the same as the time period when synMuv B mutants display open chromatin. Open chromatin during this period may allow germline genes to be poised for ectopic expression in somatic tissues of synMuv B mutants. Interestingly, the last cells to divide in the intestine are the last cells to adopt compact chromatin, suggesting that the transition to organized chromatin is coupled with differentiation. Understanding synMuv B regulation of promoter enrichment of H3K9me2 and chromatin compaction will help elucidate pathways used to achieve proper gene expression and correct development.

Strome, Susan et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Transmission and Antagonism of Germline Fate in C. elegans"

Takasaki, Teruaki, Rechtsteiner, Andreas, Gaydos, Laura, Egelhofer, Thea A, Lieb, Jason D, Petrella, Lisa, Ercan, Sevinc, Strome, Susan

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Perpetuation of the C. elegans germ line from generation to generation depends on the MES proteins, named for their maternal-effect sterile phenotype. MES-2, MES-3, and MES-6 form the worm version of the Polycomb Repressive Complex 2 and methylate histone H3 on Lys 27 (H3K27), a modification associated with repression of gene expression. MES-4 is a homolog of the mammalian NSD proteins and methylates H3K36, a modification associated with active gene expression. Numerous studies have linked MES-2/3/6 and MES-4 to global repression of gene expression from the X chromosomes in the germ line, but have also suggested that MES-4 serves an additional critical role in early primordial germ cells (PGCs). Recent chromatin immunoprecipitation analysis of the distribution of MES-4, RNA Polymerase II, and H3K36 methyl marks across the genome in early embryos suggests that MES-4 serves a memory role. In contrast to previously studied H3K36 methyltransferases, which are targeted to genes by association with Pol II, MES-4 can associate with genes in a Pol II-independent manner. The genes that MES-4 binds and methylates in embryos are genes that were previously expressed in the maternal germline, many of which are no longer expressed in embryos. These and other findings suggest that MES-4 transmits the memory of gene expression in the parental germ line to offspring and ensures the ability of the PGCs to execute a proper germline program. Our findings raise the question of how somatic cells in the embryo, which also inherit MES-4 marking of germline genes, avoid following a germline fate. The synMuv B chromatin regulators play a key role, as their loss causes somatic cells to misexpress numerous germline-specific genes and causes larvae grown at elevated temperature to arrest. Concomitant loss of maternal MES-4 suppresses the germline potential of somatic cells and larval arrest. We will discuss the opposing roles of the MES and synMuv B proteins in the germ-soma decision.

Vielle-Canonge, Anne et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Insights into chromatin organization in C. e legans from genome-wide mapping of histone modifications"

Shin, Gene, Egelhofer, Thea A, Rechtsteiner, Andreas, Vielle-Canonge, Anne, Strome, Susan, Kolasinska-Zwierz, Paulina, Latorre, Isabel, Liu, X Shirley, Ahringer, Julie A, Liu, Tao, Taing, Scott, Lieb, Jason D

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

To investigate the interplay between chromatin modifications and the regulation of nuclear processes, we are generating a genome-wide map of the locations of histones and histone tail modifications at two developmental stages (early embryo and L3), as part of the modENCODE project. Currently, we have data for >20 different epitopes at the two stages. At the level of single genes, active and repressed genes display histone modification patterns similar to those found in other organisms, e.g. H3K4me3 at the 5 ends of active genes, H3K36me3 over the bodies of active genes, and H3K9me3 over repressed genes. At the level of whole chromosomes, we observe strikingly broad domains of enrichment for several histone modifications. These domains correspond to the recombinationally defined arm and central gene-rich regions. Chromosome arms are enriched for H3K9 modifications: H3K9me2 is concentrated on both arms of each c hromosome, while H3K9me3 is especially concentrated on the arm of each chromosome containing the meiotic pairing center, as also seen by Gu and Fire (Chromosoma 2010). H3K4 modifications and other marks associated with active chromatin are enriched in the gene-rich central region of each chromosome. These broad domains are quite similar in early embryo and L3 chromatin. We will present our current maps of histone marks at the meeting and discuss the implications of our findings, that chromosomes are partitioned into central zones of largely-expressed genes and arm zones of largely-repressed genes and that events in the germline, including meiotic pairing and recombination, influence chromatin states throughout development

Ahringer, Julie et al. (2011) International Worm Meeting "A role for H4K20me1 in X chromosome dosage compensation."

Lieb, Jason, Dong, Yan, Vielle, Anne, Kotwaliwale, Chitra, Strome, Susan, Lang, Jackie, Dernburg, Abby, Rechtsteiner, Andreas, Ahringer, Julie, Ercan, Sevinc, Liu, Shirley, Liu, Tao, Dose, Andrea, Egelhofer, Thea

[International Worm Meeting, 2011]

In C. elegans, the dosage compensation complex (DCC) equalizes X chromosome gene dosage between XO males and XX hermaphrodites by repressing X transcription by two-fold specifically in hermaphrodites. The DCC is comprised of proteins homologous to the condensin complex, and it localizes to the X chromosomes in hermaphrodites but not in males. The mechanism by which the DCC downregulates expression in XX animals remains unclear. A genome-wide map of 19 histone modifications in two developmental stages of C. elegans (Liu et al, 2011) showed that the monomethylation of histone H4 at lysine 20 (H4K20me1) is enriched on the X chromosome of XX embryos and L3 larvae. H4K20me1 is also associated with actively transcribed genes on all chromosomes. The X-enrichment of H4K20me1 suggests a possible role for this modification in dosage compensation. Here, we report that H4K20me1 is enriched on the X chromosomes of XX hermaphrodites, but not in XO males. Enrichment of H4K20me1 on the X occurs after the localization of the DCC to the X and depends on the DCC function. In mammals, PR-Set7 catalyzes monomethylation of H4K20. RNAi knockdown of set-1, the likely C. elegans ortholog of PR-Set7, results in synthetic lethality with a number of dosage compensation mutations. These results support a function for H4K20me1 in dosage compensation. The X chromosome is also silenced in the germline in a DCC-independent manner. We found that in the mitotically proliferating zone of the germline, H4K20me1 is abundant on all chromosomes, but as nuclei enter meiosis, H4K20me1 levels are globally reduced. In contrast to somatic nuclei, H4K20me1 is specifically absent from the X chromosome during the meiotic prophase. H4K20me1 was recently shown to be important for chromosome condensation in mammals (Oda et al, 2009), possibly by recruiting condensin II (Liu et al, 2010). Our data indicate that the condensin-like C. elegans DCC preferentially increases H4K20me1 levels on the active X-linked genes. One plausible model for the role of H4K20me1 in dosage compensation is that preferential increase of H4K20me1 to the X may result in further recruitment of condensins to lower access of the transcription machinery to active X-linked genes. Liu et al (2011) Genome Res. 21, 227-36. Oda et al (2009) Mol Cell Biol. 29, 2278-95. Liu et al (2010) Nature. 466(7305):508-12.