Analysis of Hi-C data using SIP effectively identifies loops in organisms from C. elegans to mammals.

Chromatin loops are a major component of 3D nuclear organization, visually apparent as intense point-to-point interactions in Hi-C maps. Identification of these loops is a critical part of most Hi-C analyses. However, current methods often miss visually evident CTCF loops in Hi-C data sets from mammals, and they completely fail to identify high intensity loops in other organisms. We present SIP, Significant Interaction Peak caller, and SIPMeta, which are platform independent programs to identify and characterize these loops in a time- and memory-efficient manner. We show that SIP is resistant to noise and sequencing depth, and can be used to detect loops that were previously missed in human cells as well as loops in other organisms. SIPMeta corrects for a common visualization artifact by accounting for Manhattan distance to create average plots of Hi-C and HiChIP data. We then demonstrate that the use of SIP and SIPMeta can lead to biological insights by characterizing the contribution of several transcription factors to CTCF loop stability in human cells. We also annotate loops associated with the SMC component of the dosage compensation complex (DCC) in Caenorhabditis elegans and demonstrate that loop anchors represent bidirectional blocks for symmetrical loop extrusion. This is in contrast to the asymmetrical extrusion until unidirectional blockage by CTCF that is presumed to occur in mammals. Using HiChIP and multiway ligation events, we then show that DCC loops form a network of strong interactions that may contribute to X Chromosome-wide condensation in C. elegans hermaphrodites.

Condensin I protects meiotic cohesin from WAPL-1 mediated removal.

Condensin complexes are key determinants of higher-order chromatin structure and are required for mitotic and meiotic chromosome compaction and segregation. We identified a new role for condensin in the maintenance of sister chromatid cohesion during C. elegans meiosis. Using conventional and stimulated emission depletion (STED) microscopy we show that levels of chromosomally-bound cohesin were significantly reduced in dpy-28 mutants, which lack a subunit of condensin I. SYP-1, a component of the synaptonemal complex central region, was also diminished, but no decrease in the axial element protein HTP-3 was observed. Surprisingly, the two key meiotic cohesin complexes of C. elegans were both depleted from meiotic chromosomes following the loss of condensin I, and disrupting condensin I in cohesin mutants increased the frequency of detached sister chromatids. During mitosis and meiosis in many organisms, establishment of cohesion is antagonized by cohesin removal by Wapl, and we found that condensin I binds to C. elegans WAPL-1 and counteracts WAPL-1-dependent cohesin removal. Our data suggest that condensin I opposes WAPL-1 to promote stable binding of cohesin to meiotic chromosomes, thereby ensuring linkages between sister chromatids in early meiosis.

Anchoring of Heterochromatin to the Nuclear Lamina Reinforces Dosage Compensation-Mediated Gene Repression.

Higher order chromosome structure and nuclear architecture can have profound effects on gene regulation. We analyzed how compartmentalizing the genome by tethering heterochromatic regions to the nuclear lamina affects dosage compensation in the nematode C. elegans. In this organism, the dosage compensation complex (DCC) binds both X chromosomes of hermaphrodites to repress transcription two-fold, thus balancing gene expression between XX hermaphrodites and XO males. X chromosome structure is disrupted by mutations in DCC subunits. Using X chromosome paint fluorescence microscopy, we found that X chromosome structure and subnuclear localization are also disrupted when the mechanisms that anchor heterochromatin to the nuclear lamina are defective. Strikingly, the heterochromatic left end of the X chromosome is less affected than the gene-rich middle region, which lacks heterochromatic anchors. These changes in X chromosome structure and subnuclear localization are accompanied by small, but significant levels of derepression of X-linked genes as measured by RNA-seq, without any observable defects in DCC localization and DCC-mediated changes in histone modifications. We propose a model in which heterochromatic tethers on the left arm of the X cooperate with the DCC to compact and peripherally relocate the X chromosomes, contributing to gene repression.

The onset of C. elegans dosage compensation is linked to the loss of developmental plasticity.

Dosage compensation (DC) equalizes X-linked gene expression between sexes. In Caenorhabditis elegans, the dosage compensation complex (DCC) localizes to both X chromosomes in hermaphrodites and downregulates gene expression 2-fold. The DCC first localizes to hermaphrodite X chromosomes at the 30-cell stage, coincident with a developmental transition from plasticity to differentiation. To test whether DC onset is linked to loss of developmental plasticity, we established a timeline for the accumulation of DC-mediated chromatin features on X (depletion of histone H4 lysine 16 acetylation (H4K16ac) and enrichment of H4K20 monomethylation (H4K20me1)) in both wild type and developmentally delayed embryos. Surprisingly, we found that H4K16ac is depleted from the X even before the 30-cell stage in a DCC-independent manner. This depletion requires the activities of MES-2, MES-3, and MES-6 (a complex similar to the Polycomb Repressive Complex 2), and MES-4. By contrast, H4K20me1 becomes enriched on X chromosomes several cell cycles after DCC localization to the X, suggesting that it is a late mark in DC. MES-2 also promotes differentiation, and mes-2 mutant embryos exhibit prolonged developmental plasticity. Consistent with the hypothesis that the onset of DC is linked to differentiation, DCC localization and H4K20me1 accumulation on the X chromosomes are delayed in mes mutant hermaphrodite embryos. Furthermore, the onset of hermaphrodite-specific transcription of sdc-2 (which triggers DC) is delayed in mes-2 mutants. We propose that as embryonic blastomeres lose their developmental plasticity, hermaphrodite X chromosomes transition from a MES protein-regulated state to DCC-mediated repression.

Biofilm formation protects Escherichia coli against killing by Caenorhabditis elegans and Myxococcus xanthus.

Enteric bacteria, such as Escherichia coli, are exposed to a variety of stresses in the nonhost environment. The development of biofilms provides E. coli with resistance to environmental insults, such as desiccation and bleach. We found that biofilm formation, specifically production of the matrix components curli and cellulose, protected E. coli against killing by the soil-dwelling nematode Caenorhabditis elegans and the predatory bacterium Myxococcus xanthus. Additionally, matrix-encased bacteria at the air-biofilm interface exhibited ∼40-fold-increased survival after C. elegans and M. xanthus killing compared to the non-matrix-encased cells that populate the interior of the biofilm. To determine if nonhost Enterobacteriaceae reservoirs supported biofilm formation, we grew E. coli on media composed of pig dung or commonly contaminated foods, such as beef, chicken, and spinach. Each of these medium types provided a nutritional environment that supported matrix production and biofilm formation. Altogether, we showed that common, nonhost reservoirs of E. coli supported the formation of biofilms that subsequently protected E. coli against predation.

Different roles for Aurora B in condensin targeting during mitosis and meiosis.

Condensin complexes are essential for mitotic and meiotic chromosome segregation. Caenorhabditis elegans, like other metazoans, has two distinct mitotic and meiotic condensin complexes (I and II), which occupy distinct chromosomal domains and perform non-redundant functions. Despite the differences in mitotic and meiotic chromosome behavior, we uncovered several conserved aspects of condensin targeting during these processes. During both mitosis and meiosis, condensin II loads onto chromosomes in early prophase, and condensin I loads at entry into prometaphase. During both mitosis and meiosis, the localization of condensin I, but not condensin II, closely parallels the localization of the chromosomal passenger kinase Aurora B (AIR-2 in C. elegans). Interestingly, condensin I and AIR-2 also colocalize on the spindle midzone during anaphase of mitosis, and between separating chromosomes during anaphase of meiosis. Consistently, AIR-2 affects the targeting of condensin I but not condensin II. However, the role AIR-2 plays in condensin I targeting during these processes is different. In mitosis, AIR-2 activity is required for chromosomal association of condensin I. By contrast, during meiosis, AIR-2 is not required for condensin I chromosomal association, but it provides cues for correct spatial targeting of the complex.

xol-1, the master sex-switch gene in C. elegans, is a transcriptional target of the terminal sex-determining factor TRA-1.

In the nematode Caenorhabditis elegans, sex is determined by the ratio of X chromosomes to sets of autosomes: XX animals (2X:2A=1.0) develop as hermaphrodites and XO animals (1X:2A=0.5) develop as males. TRA-1, the worm ortholog of Drosophila Cubitus interruptus and mammalian Gli (Glioma-associated homolog) proteins, is the terminal transcription factor of the C. elegans sex-determination pathway, which specifies hermaphrodite fate by repressing male-specific genes. Here we identify a consensus TRA-1 binding site in the regulatory region of xol-1, the master switch gene controlling sex determination and dosage compensation. xol-1 is normally expressed in males, where it promotes male development and prevents dosage compensation. We show that TRA-1 binds to the consensus site in the xol-1 promoter in vitro and inhibits the expression of xol-1 in XX animals in vivo. Furthermore, inactivation of tra-1 enhances, whereas hyperactivation of tra-1 suppresses, lethality in animals with elevated xol-1 activity. These data imply the existence of a regulatory feedback loop within the C. elegans sex-determination and dosage-compensation cascade that ensures the accurate dose of X-linked genes in cells destined to adopt hermaphrodite fate.

Restricting dosage compensation complex binding to the X chromosomes by H2A.Z/HTZ-1.

Dosage compensation ensures similar levels of X-linked gene products in males (XY or XO) and females (XX), despite their different numbers of X chromosomes. In mammals, flies, and worms, dosage compensation is mediated by a specialized machinery that localizes to one or both of the X chromosomes in one sex resulting in a change in gene expression from the affected X chromosome(s). In mammals and flies, dosage compensation is associated with specific histone posttranslational modifications and replacement with variant histones. Until now, no specific histone modifications or histone variants have been implicated in Caenorhabditis elegans dosage compensation. Taking a candidate approach, we have looked at specific histone modifications and variants on the C. elegans dosage compensated X chromosomes. Using RNAi-based assays, we show that reducing levels of the histone H2A variant, H2A.Z (HTZ-1 in C. elegans), leads to partial disruption of dosage compensation. By immunofluorescence, we have observed that HTZ-1 is under-represented on the dosage compensated X chromosomes, but not on the non-dosage compensated male X chromosome. We find that reduction of HTZ-1 levels by RNA interference (RNAi) and mutation results in only a very modest change in dosage compensation complex protein levels. However, in these animals, the X chromosome-specific localization of the complex is partially disrupted, with some nuclei displaying DCC localization beyond the X chromosome territory. We propose a model in which HTZ-1, directly or indirectly, serves to restrict the dosage compensation complex to the X chromosome by acting as or regulating the activity of an autosomal repellant.

Dual roles for nuclear RNAi Argonautes in Caenorhabditis elegans dosage compensation.

Dosage compensation involves chromosome-wide gene regulatory mechanisms which impact higher order chromatin structure and are crucial for organismal health. Using a genetic approach, we identified Argonaute genes which promote dosage compensation in Caenorhabditis elegans. Dosage compensation in C. elegans hermaphrodites is initiated by the silencing of xol-1 and subsequent activation of the dosage compensation complex which binds to both hermaphrodite X chromosomes and reduces transcriptional output by half. A hallmark phenotype of dosage compensation mutants is decondensation of the X chromosomes. We characterized this phenotype in Argonaute mutants using X chromosome paint probes and fluorescence microscopy. We found that while nuclear Argonaute mutants hrde-1 and nrde-3, as well as mutants for the piRNA Argonaute prg-1, exhibit derepression of xol-1 transcripts, they also affect X chromosome condensation in a xol-1-independent manner. We also characterized the physiological contribution of Argonaute genes to dosage compensation using genetic assays and found that hrde-1 and nrde-3 contribute to healthy dosage compensation both upstream and downstream of xol-1.

The worm solution: a chromosome-full of condensin helps gene expression go down.

Dosage compensation in the nematode Caenorhabditis elegans is achieved by the binding of a condensin-like dosage compensation complex (DCC) to both X chromosomes in hermaphrodites to downregulate gene expression two-fold. Condensin I(DC), a sub-part of the DCC, differs from the mitotic condensin I complex by a single subunit, strengthening the connection between dosage compensation and mitotic chromosome condensation. The DCC is targeted to X chromosomes by initial binding to a number of recruiting elements, followed by dispersal or spreading to secondary sites. While the complex is greatly enriched on the X chromosomes, many sites on autosomes also bind the complex. DCC binding does not correlate with DCC-mediated repression, suggesting that the complex acts in a chromosome-wide manner, rather than on a gene-by-gene basis. Worm dosage compensation represents an excellent model system to study how condensin-mediated changes in higher order chromatin organization affect gene expression.

A deletion at the mouse Xist gene exposes trans-effects that alter the heterochromatin of the inactive X chromosome and the replication time and DNA stability of both X chromosomes.

The inactive X chromosome of female mammals displays several properties of heterochromatin including late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, and methylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in female mouse fibroblasts led to the appearance of two histone modifications throughout the inactive X chromosome usually associated with euchromatin: histone H4 acetylation and histone H3 lysine-4 methylation. Despite these euchromatic properties, the inactive X chromosome was replicated even later in S phase than in wild-type female cells. Homozygosity for the deletion also caused regions of the active X chromosome that are associated with very high concentrations of LINE-1 elements to be replicated very late in S phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNA of both X chromosomes, leading to deletions and translocations. This was accompanied by the phosphorylation of p53 at serine-15, an event that occurs in response to DNA damage, and the accumulation of gamma-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains the DNA stability of both X chromosomes.

MORC-1 Integrates Nuclear RNAi and Transgenerational Chromatin Architecture to Promote Germline Immortality.

Germline-expressed endogenous small interfering RNAs (endo-siRNAs) transmit multigenerational epigenetic information to ensure fertility in subsequent generations. In Caenorhabditis elegans, nuclear RNAi ensures robust inheritance of endo-siRNAs and deposition of repressive H3K9me3 marks at target loci. How target silencing is maintained in subsequent generations is poorly understood. We discovered that morc-1 is essential for transgenerational fertility and acts as an effector of endo-siRNAs. Unexpectedly, morc-1 is dispensable for siRNA inheritance but is required for target silencing and maintenance of siRNA-dependent chromatin organization. A forward genetic screen identified mutations in met-1, which encodes an H3K36 methyltransferase, as potent suppressors of morc-1(-) and nuclear RNAi mutant phenotypes. Further analysis of nuclear RNAi and morc-1(-) mutants revealed a progressive, met-1-dependent enrichment of H3K36me3, suggesting that robust fertility requires repression of MET-1 activity at nuclear RNAi targets. Without MORC-1 and nuclear RNAi, MET-1-mediated encroachment of euchromatin leads to detrimental decondensation of germline chromatin and germline mortality.

An H4K16 histone acetyltransferase mediates decondensation of the X chromosome in C. elegans males.

BACKGROUND: In C. elegans, in order to equalize gene expression between the sexes and balance X and autosomal expression, two steps are believed to be required. First, an unknown mechanism is hypothesized to upregulate the X chromosome in both sexes. This mechanism balances the X to autosomal expression in males, but creates X overexpression in hermaphrodites. Therefore, to restore the balance, hermaphrodites downregulate gene expression twofold on both X chromosomes. While many studies have focused on X chromosome downregulation, the mechanism of X upregulation is not known. RESULTS: To gain more insight into X upregulation, we studied the effects of chromatin condensation and histone acetylation on gene expression levels in male C. elegans. We have found that the H4K16 histone acetyltransferase MYS-1/Tip60 mediates dramatic decondensation of the male X chromosome as measured by FISH. However, RNA-seq analysis revealed that MYS-1 contributes only slightly to upregulation of gene expression on the X chromosome. These results suggest that the level of chromosome decondensation does not necessarily correlate with the degree of gene expression change in vivo. Furthermore, the X chromosome is more sensitive to MYS-1-mediated decondensation than the autosomes, despite similar levels of H4K16ac on all chromosomes, as measured by ChIP-seq. H4K16ac levels weakly correlate with gene expression levels on both the X and the autosomes, but highly expressed genes on the X chromosome do not contain exceptionally high levels of H4K16ac. CONCLUSION: These results indicate that H4K16ac and chromosome decondensation influence regulation of the male X chromosome; however, they do not fully account for the high levels of gene expression observed on the X chromosomes.

Balancing up and downregulation of the C. elegans X chromosomes.

In Caenorhabditis elegans, males have one X chromosome and hermaphrodites have two. Emerging evidence indicates that the male X is transcriptionally more active than autosomes to balance the single X to two sets of autosomes. Because upregulation is not limited to males, hermaphrodites need to strike back and downregulate expression from the two X chromosomes to balance gene expression in their genome. Hermaphrodite-specific downregulation involves binding of the dosage compensation complex to both Xs. Advances in recent years revealed that the action of the dosage compensation complex results in compaction of the X chromosomes, changes in the distribution of histone modifications, and ultimately limiting RNA Polymerase II loading to achieve chromosome-wide gene repression.

Condensin-mediated chromosome organization and gene regulation.

In many organisms sexual fate is determined by a chromosome-based method which entails a difference in sex chromosome-linked gene dosage. Consequently, a gene regulatory mechanism called dosage compensation equalizes X-linked gene expression between the sexes. Dosage compensation initiates as cells transition from pluripotency to differentiation. In Caenorhabditis elegans, dosage compensation is achieved by the dosage compensation complex (DCC) binding to both X chromosomes in hermaphrodites to downregulate gene expression by twofold. The DCC contains a subcomplex (condensin I(DC)) similar to the evolutionarily conserved condensin complexes which play a fundamental role in chromosome dynamics during mitosis. Therefore, mechanisms related to mitotic chromosome condensation are hypothesized to mediate dosage compensation. Consistent with this hypothesis, monomethylation of histone H4 lysine 20 is increased, whereas acetylation of histone H4 lysine 16 is decreased, both on mitotic chromosomes and on interphase dosage compensated X chromosomes in worms. These observations suggest that interphase dosage compensated X chromosomes maintain some characteristics associated with condensed mitotic chromosome. This chromosome state is stably propagated from one cell generation to the next. In this review we will speculate on how the biochemical activities of condensin can achieve both mitotic chromosome compaction and gene repression.

The C. elegans dosage compensation complex mediates interphase X chromosome compaction.

BACKGROUND: Dosage compensation is a specialized gene regulatory mechanism which equalizes X-linked gene expression between sexes. In Caenorhabditis elegans, dosage compensation is achieved by the activity of the dosage compensation complex (DCC). The DCC localizes to both X chromosomes in hermaphrodites to downregulate gene expression by half. The DCC contains a subcomplex (condensin I(DC)) similar to the evolutionarily conserved condensin complexes which play fundamental roles in chromosome dynamics during mitosis and meiosis. Therefore, mechanisms related to mitotic chromosome condensation have been long hypothesized to mediate dosage compensation. However experimental evidence was lacking. RESULTS: Using 3D FISH microscopy to measure the volumes of X and chromosome I territories and to measure distances between individual loci, we show that hermaphrodite worms deficient in DCC proteins have enlarged interphase X chromosomes when compared to wild type. By contrast, chromosome I is unaffected. Interestingly, hermaphrodite worms depleted of condensin I or II show no phenotype. Therefore X chromosome compaction is specific to condensin I(DC). In addition, we show that SET-1, SET-4, and SIR-2.1, histone modifiers whose activity is regulated by the DCC, need to be present for the compaction of the X chromosome territory. CONCLUSION: These results support the idea that condensin I(DC), and the histone modifications regulated by the DCC, mediate interphase X chromosome compaction. Our results link condensin-mediated chromosome compaction, an activity connected to mitotic chromosome condensation, to chromosome-wide repression of gene expression in interphase.

Pluripotent cells will not dosage compensate.

Dosage compensation is the mechanism that balances gene expression levels between males and females as well as between the X chromosome and autosomes. In mammals, loss of pluripotency and differentiation are closely linked with the onset of dosage compensation. Pluripotency factors negatively regulate Xist (the non-coding RNA that triggers X chromosome inactivation) and positively regulate Tsix, a repressor of Xist, to inhibit dosage compensation. In addition, X chromosome dose also regulates exit from the pluripotent state. A double dose of X chromosomes in undifferentiated female cells inhibits the MAPK and Gsk3 signaling pathways and activates the Akt pathway, thereby blocking differentiation. Here we review our recent report, which showed that the onset of dosage compensation is also linked to the loss of pluripotency in C. elegans. We discuss these findings in light of what is known about pluripotency and differentiation in this organism.

Condensin and the spindle midzone prevent cytokinesis failure induced by chromatin bridges in C. elegans embryos.

BACKGROUND: During cell division, chromosomes must clear the path of the cleavage furrow before the onset of cytokinesis. The abscission checkpoint in mammalian cells stabilizes the cleavage furrow in the presence of a chromatin obstruction. This provides time to resolve the obstruction before the cleavage furrow regresses or breaks the chromosomes, preventing aneuploidy or DNA damage. Two unanswered questions in the proposed mechanistic pathway of the abscission checkpoint concern factors involved in (1) resolving the obstructions and (2) coordinating obstruction resolution with the delay in cytokinesis. RESULTS: We found that the one-cell and two-cell C. elegans embryos suppress furrow regression following depletion of essential chromosome-segregation factors: topoisomerase II(TOP-2), CENP-A(HCP-3), cohesin, and to a lesser degree, condensin. Chromatin obstructions activated Aurora B(AIR-2) at the spindle midzone, which is needed for the abscission checkpoint in other systems. Condensin I, but not condensin II, localizes to the spindle midzone in anaphase and to the midbody during normal cytokinesis. Interestingly, condensin I is enriched on chromatin bridges and near the midzone/midbody in an AIR-2-dependent manner. Disruption of AIR-2, the spindle midzone, or condensin leads to cytokinesis failure in a chromatin-obstruction-dependent manner. Examination of the condensin-deficient embryos uncovered defects in both the resolution of the chromatin obstructions and the maintenance of the stable cleavage furrow. CONCLUSIONS: We postulate that condensin I is recruited by Aurora B(AIR-2) to aid in the resolution of chromatin obstructions and also helps generate a signal to maintain the delay in cytokinesis.

H3K56me3 is a novel, conserved heterochromatic mark that largely but not completely overlaps with H3K9me3 in both regulation and localization.

Histone lysine (K) methylation has been shown to play a fundamental role in modulating chromatin architecture and regulation of gene expression. Here we report on the identification of histone H3K56, located at the pivotal, nucleosome DNA entry/exit point, as a novel methylation site that is evolutionary conserved. We identify trimethylation of H3K56 (H3K56me3) as a modification that is present during all cell cycle phases, with the exception of S-phase, where it is underrepresented on chromatin. H3K56me3 is a novel heterochromatin mark, since it is enriched at pericentromeres but not telomeres and is thereby similar, but not identical, to the localization of H3K9me3 and H4K20me3. Possibly due to H3 sequence similarities, Suv39h enzymes, responsible for trimethylation of H3K9, also affect methylation of H3K56. Similarly, we demonstrate that trimethylation of H3K56 is removed by members of the JMJD2 family of demethylases that also target H3K9me3. Furthermore, we identify and characterize mouse mJmjd2E and its human homolog hKDM4L as novel, functionally active enzymes that catalyze the removal of two methyl groups from trimethylated H3K9 and K56. H3K56me3 is also found in C. elegans, where it co-localizes with H3K9me3 in most, but not all, tissues. Taken together, our findings raise interesting questions regarding how methylation of H3K9 and H3K56 is regulated in different organisms and their functional roles in heterochromatin formation and/or maintenance.