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[
International Worm Meeting,
2007]
Animals with chromosome-based mechanisms of sex determination must equalize expression of X-linked genes between the sexes. In C. elegans, a dosage compensation complex (DCC) binds specifically to each X in XX hermaphrodites to halve transcription, matching the X-linked expression observed in XO males. We recently mapped the genomic binding location of two DCC components, DPY-27 and SDC-3, by chromatin immunoprecipitation followed by whole-genome tiling microarrays (ChIP-chip; Ercan et al, Nat Genet. 39(3):403-8.) We found that strong foci of DCC binding were observed on X, surrounded by broader, more uniform regions of DCC localization. Binding foci, but not adjacent regions of localization, were distinguished by clusters of a stereotypic 10-bp DNA sequence, suggesting a recruitment-and-spreading mechanism for X recognition. We suggested that DNA sequence and chromatin signals cooperate to target the DCC to the promoters of individual genes. We further suggested that this recruitment, which is tuned to the level of expression, may be followed by a short-range polymerase-coupled spreading mechanism. We hope to report the results of some follow-up experiments we have initiated to test these models of X-chromosome recruitment and spreading. In addition, we are initiating new genome-wide studies of chromatin dynamics during C. elegans embryogenesis, and anticipate presenting our initial results at the meeting.
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[
International Worm Meeting,
2011]
Animals with different numbers of X chromosomes in males and females possess mechanisms to compensate for the difference in the X-linked gene dose between the two sexes. In addition to the X chromosome dose difference between the sexes, the presence of a single-copy X chromosome per two-copy diploid autosomes creates an important problem for males, because all X-linked genes are haploinsufficient compared to the autosomal genes. In C. elegans, hermaphrodites (XX) contain two Xes, whereas males (XO) contain a single X, therefore facing X haploinsufficiency. By performing microarray analysis of RNA abundance in XX and XO worms, we observed that the overall transcript levels from the X chromosome in both XX and XO animals is similar to that of overall expression from autosomes. This suggests that transcription from the single X in XO L3 hermaphrodites (TY2205,
her-1(
e1520)
sdc-3(
y126) V;
xol-1(
y9) X) is increased approximately two-fold. The mechanism of this upregulation is unclear. We had shown that the X chromosome promoters have higher GC content compared to the autosomes (Ercan et al 2010), suggesting a DNA-encoded mechanism of transcriptional regulation. We will study X upregulation by comparing transcription of orthologus genes that are on the X versus autosomes in four Caenorhabditis species. Ercan S, Lubling Y, Segal E, Lieb JD. High nucleosome occupancy is encoded at X-linked gene promoters in C. elegans. Genome Res. 2011 Feb;21(2):237-44. PMID:21177966.
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[
International Worm Meeting,
2015]
Specificity of transcription factor (TF) targeting is at the heart of gene regulation, but it is a process that is not well understood. TF binding specificity is accomplished in part by the chromatin accessibility of the DNA sequence motifs to which the TF binds. To understand X-specific binding of the dosage compensation complex (DCC), we will map chromatin accessibility across the genome. This will allow us to test whether the 10 base pair DNA sequence motif that is important for DCC recruitment is more accessible on the X. We use transposase-accessible chromatin sequencing (ATAC-seq) to map chromatin accessibility. In this technique, transposases insert sequencing adaptors into regions of chromatin not occupied by nucleosomes. ATAC-seq has been developed by the Greenleaf lab using human lymphoblastoid cells. We have obtained preliminary data from embryo and L3 larval stage worms and naked DNA as a control. Our results indicate that the transposase has a sequence preference for insertion. In embyros, we do not see many distinct peaks, but we do see those in L3s. Currently, we are working to optimize ATAC-seq in C. elegans for embryo and larval stages. .
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[
International Worm Meeting,
2013]
The genetic networks that direct embryonic development rely on specific levels of gene expression and so do not tolerate large changes in gene copy number, also known as gene dose. Despite having different numbers of X chromosomes, male and female embryos develop similarly. This is due to dosage compensation mechanisms that regulate X chromosome transcription during embryogenesis. These are well studied during later development and adulthood but the mechanisms of dosage compensation during early embryogenesis remain unclear. In C. elegans, imprinted silencing of the paternally contributed X chromosome provides a potential mechanism for early embryo dosage compensation. To determine whether chromosome wide dosage compensation occurs prior to activation of the canonical Dosage Compensation Complex (DCC), populations of hermaphrodite and mixed sex of embryos younger than 40-cell stage were collected their transcriptomes were sequenced. Expression levels from the X chromosome are higher in hermaphrodites, suggesting that dosage compensation does not occur uniformly across the entire X chromosome in the very early embryo. In order to determine whether paternal X inactivation acts selectively on specific genes, we have begun to analyze expression of paternally contributed alleles in hybrid polymorphic embryos. Expression of paternal SNPs in the early embryo will allow us to determine which X linked genes are expressed from the paternal X chromosome and which genes are expressed solely due to maternally contributed mRNA and/or expression from the maternally contributed X chromosome.
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[
International Worm Meeting,
2015]
Sequence-specific transcription factors (TFs) bind to a small fraction of their sequence motifs in the genome. Previous studies showed that strength and number of DNA sequence motifs, cooperative binding, and chromatin structure influence TF specificity. Here, we studied X-chromosome specific targeting of a specialized condensin complex that constitutes the core of the C. elegans dosage compensation complex. Based on the ChIP-seq analysis of DCC recruiters (SDC-2, SDC-3, DPY-30), we have categorized condensin DC recruitment motifs into "bound" and "unbound". Some of the unbound motifs have the same characteristics (motif strength and clustering) as bound motifs and were termed "potential". Using these categories we tried to distinguish bound and potential motif containing sites using motif strength and number, histone modifications, chromatin accessibility, and genomic distribution of the motifs. Our preliminary data suggest that histone modifications do not explain X-specificity of condensin DC recruitment. Motif strength and number, as well as the intergenic distribution of the motifs explain part of the specificity, but not all. We also noted that while condensin DC recruiters localize to hundreds of sites on the autosomes, condensin DC-specific subunit DPY-27 is not recruited to those sites. It is possible that some unknown feature of the X chromosome is important for the X-specific recruitment of condensin DC.
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[
International Worm Meeting,
2021]
Techniques to study protein function in living cells is useful in the investigation of biological regulatory mechanisms. Among the Caenorhabditis elegans transgenic toolkits, the Auxin-Inducible Degradation (AID) system provides spatial and temporal control of protein degradation in both a conditional and reversible manner. The AID system utilizes the F-box related protein TIR1, which upon auxin addition, leads to the degradation of AID degron tagged target protein. In C. elegans, the usage of AID system is commonly in transgenic lines that express the modified Arabidopsis thaliana TIR1. Here, we report that lifelong exposure to 0.01mM, 0.1mM, and 1mM auxin does not significantly affect the morphological quantified phenotypic features or embryo lethality in TIR1 expressing strain CA1200. However, Pol-II ChIP-seq enrichment profile upon 60 minutes of auxin treatment in CA1200 worms shows a dramatic shift within coding regions, which is notable for researchers who are using this strain doing any ChIP-seq analysis.
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[
International Worm Meeting,
2021]
Chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) has been the gold standard for analyzing interactions between DNA and proteins in the field of gene regulation. It does, however, require a considerable number of cells. Alternative strategies like CUT&RUN and CUT&Tag have been introduced to overcome the limitations of cell number. CUT&Tag utilizes a transposome consisting of a Tn5 transposase fused with proteins A and G, which direct the transposase to an antibody bound to its target. The transposase is also pre-loaded with sequencing adapters, which allows for antibody directed tagmentation followed by library preparation. Here, we applied CUT&Tag to purified nuclei of embryos using at least five different antibodies. We will present our progress in applying this technique to nematodes and share our data.
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[
International Worm Meeting,
2013]
In Caenorhabditis species, dosage compensation acts to reduce transcriptional output of both female/hermaphrodite X chromosomes by one half. This balances X expression between the sexes, but potentially gives females the same problem faced by males: functional monosomy of the X chromosome. It has been proposed that a mechanism evolved to upregulate X expression to balance X and autosomal transcription, thereby overcoming male monosomy. However, owing to biased gene content and tissue-specific regulation of the X, direct comparison of X and autosomal transcription is difficult. In order to more directly compare X and autosomal transcription we looked at expression of 1:1 orthologs that are differentially located on the X or an autosome between two nematode species. Our work focused on four species: C. elegans, C. briggsae, C. remanei, and Pristionchus pacificus. The C. elegans and C. briggsae genomes are well assembled and annotated. The genomes of C. remanei and P. pacificus have been sequenced, but their genes have not yet been assigned to chromosomal locations. Since our analysis depends on comparing differentially located orthologs, we first needed to map genes to either the X or autosomes. We took a read-depth-variation approach. We performed genomic DNA-seq in males and females/hermaphrodites of C. brenneri, C. remanei and P. pacificus. C. briggsae males and hermaphrodites were also sequenced as controls. Genes located on the X chromosome were expected to have a 1:2 ratio of sequencing coverage between males and hermaphrodites (X:XX) and all autosomal genes a 1:1 ratio. Our analysis yielded a list of X and autosomal genes for each of the four nematode species and allowed the identification of differentially located 1:1 orthologs. Comparison of X and autosomal transcription showed no bias towards male upregulation of X-located orthologs.
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[
International Worm Meeting,
2015]
Multi-subunit condensin complexes are essential for chromosome condensation during both mitosis and meiosis and have also been implicated as having important roles in transcriptional regulation during interphase. Metazoans contain two condensin complexes, I and II, which specifically localize to different chromosome regions where they perform different functions. Caenorhabditis elegans contains a third condensin complex, Condensin DC, whose localization is uniquely restricted to the hermaphrodite X chromosome where it acts as part of the Dosage Compensation Complex (DCC) to repress X-transcription. The regulatory mechanisms by which Condensin DC is targeted specifically to the X chromosome are not yet fully understood.As part of the DCC, Condensin DC interacts with at least four other non-condensin proteins, including two zinc-finger-containing proteins, SDC-2 and SDC-3, which act to recruit Condensin DC to approximately 100 recruitment sites across the X-chromosome. Evidence suggests that this initial targeting is sequence-dependent. Sites of initial recruitment, termed recruitment elements on X (rex), are enriched for a 10bp recruitment motif. Our analyses indicate that this motif is four times enriched on the X-chromosome as compared to autosomes and is often clustered at rex-sites. However, the motif is not unique to the X-chromosome; both the X chromosome and the autosomes contain many perfect matches that are not bound by the DCC. Further, we show that insertion of a rex-site in single-copy onto an autosome fails to detectably recruit DCC. Increasing the number of inserted rex-site copies overcomes the inability recruit on autosomes. We conclude that motif sequence, while important for DCC recruitment, is not sufficient to recruit the complex on its own. We hypothesize that chromosomal context of the X chromosome facilitates the specificity of DCC recruitment. .
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[
International Worm Meeting,
2015]
Condensins are evolutionarily conserved protein complexes that regulate chromosome structure in eukaryotes. However, the mechanisms of condensin binding remain unknown. In C. elegans, a condensin complex known as the dosage compensation complex (DCC) specifically binds to both X chromosomes in hermaphrodites and represses transcription by half to equalize hermaphrodite (XX) and male (XO) X-expression. The DCC serves as a clear paradigm to study the mechanisms of condensin binding because we can distinguish two modes of binding: recruitment and spreading. Recruitment is fairly well understood, but spreading is not. Our ChIP-seq analysis of DPY-27 binding in embryos, L3, and young adult stages indicate that DCC binding correlates positively with active transcription. Here, using X;A fusion strains, we show that DPY-27 spreading drops off at regions with lower transcription. To test if DCC spreading is facilitated by RNA Pol II, we will target a catalytically inactive Cas9 protein (dCas9) to block RNA Pol II, and determine how this affects DCC spreading. Determining the mechanisms of condensin spreading is important for understanding how condensins carry out their function on a chromosome wide-basis.