Barbara Meyer (2008) Development & Evolution Meeting "Gametogenesis, Meiosis, & Sex Determination"

Barbara Meyer

[Development & Evolution Meeting, 2008]

No abstract submitted

Barbara Meyer et al. (2006) Development & Evolution Meeting "Chromosome Repression and Segregation."

Barbara Meyer, Aaron Severson, Judith Jans, David Mets, Ray Chan, Patrick McDonel

[Development & Evolution Meeting, 2006]

Chromosomes must be properly expressed, resolved, compacted, and segregated for genome stability. These diverse processes are controlled by an interacting set of proteins and complexes. On set of such proteins, the condensin complex, is essential for chromosome resolution and compaction during mitosis and meiosis. A homologous set of proteins is essential for the X-chromosome-wide process of dosage compensation, which ensures that males (XO) and hermaphrodites (XX) express equal levels of X-linked gene products, despite their difference in X chromosome dose. This dosage compensation complex (DCC) binds the entirety of both hermaphrodite X chromosomes to achieve chromosome-wide reduction in gene expression. The DCC not only resembles mitotic condensin, it shares a component with condensin, and DCC components also participate in other aspects of chromosome segregation, for example the regulation of crossover interference during meiosis. This talk will focus on the connection between dosage compensation and chromosome segregation and on the mechanism by which the DCC specifically recognizes and binds X chromosomes.

Pferdehirt, Rebecca et al. (2009) International Worm Meeting "Targeting the Condensin-like Dosage Compensation Complex to X."

Pferdehirt, Rebecca, Meyer, Barbara

[International Worm Meeting, 2009]

X-chromosome dosage compensation is a chromosome-wide regulatory mechanism that equalizes levels of X gene products between males (1X) and females (2X). In C. elegans, dosage compensation is executed by a multi-protein complex that resembles condensin, a conserved complex involved in chromosome compaction, resolution and segregation. This dosage compensation complex (DCC) binds both X chromosomes of XX animals to decrease X transcript levels by about two-fold. The mechanisms by which the DCC is targeted to X are not well understood. Our recent work has revealed that multiple inputs are important for orchestrating proper DCC localization and function, ranging from DNA sequence motifs to post-translational protein modification. The DCC binds to two types of sites on X. rex sites recruit the DCC in an autonomous manner using a 12 bp consensus motif that is enriched on X, is clustered within rex sites, and is critical for DCC binding. dox sites fail to bind the DCC when detached from X, lack the X-enriched motif variants and, unlike rex sites, are enriched in promoters of genes. ChIP-chip of the DCC in embryos mutant for other DCC components has revealed differential requirements for binding rex versus dox sites and for loading of specific DCC subunits. These results refine our understanding of the hierarchy of DCC assembly and binding. We have discovered that post-translational modification by the small ubiquitin-like molecule SUMO (smo-1 in C. elegans) is essential for proper targeting of the DCC to X. Reduction of sumoylation by RNAi causes a redistribution of DCC binding from X to autosomes such that DCC binding sites are significantly reduced on X and increased on autosomes. Although all DCC components tested show defects in binding by ChIP-chip, they do not all behave in precisely the same manner with respect to rex and dox site binding, suggesting sumoylation may be important for proper assembly of the DCC on X. smo-1 RNAi also causes significant over-expression of genes on X, as measured by expression microarrays. Finally, preliminary data suggests that one or more members of the DCC are targets of sumoylation. Together these results indicate that sumoylation is one of multiple inputs required for proper targeting of DCC assembly on X. Sumoylation, perhaps of one or more DCC components, may direct DCC localization by either repelling the DCC from autosomes or by actively directing its preferential binding to X. Dosage compensation requires cumulative action of multiple discrete, yet common mechanisms to direct sex-specific localization and function of a universal complex involved in genome-wide chromosome dynamics.

Fuda, Nicholas et al. (2015) International Worm Meeting "The role of general transcription factors in dosage compensation."

Fuda, Nicholas, Meyer, Barbara

[International Worm Meeting, 2015]

X chromosome gene expression is equalized between the sexes in dosage compensation by reducing transcription on the hermaphrodite X chromosome through a specialized condensin complex. Recently, our lab used Global Run-On sequencing (GRO-seq) to identify the transcription start sites (TSS) for 6353 genes and show that dosage compensation reduces X chromosome transcription by decreasing RNA polymerase II (Pol II) recruitment or initiation. The mechanisms behind this regulation are still unclear, and little research has been conducted into the basic transcriptional mechanisms in the worm. Extensive studies from various model organisms have identified the general transcription factors (GTFs) required for Pol II recruitment and formation of the pre-initiation complex (PIC). These studies have defined an ordered assembly of GTFs. TATA Binding Protein interacts with its promoter sequence element and interacts with TFIIIA, TFIIB, and TFIID. These factors recruit TFIIF and Pol II to the promoter. Then, TFIIE and TFIIH are recruited and aid in promoter melting and transcription initiation. In addition, the Mediator complex interacts with Pol II and enhances transcription. GTF binding on promoters can provide insight to the steps in Pol II recruitment impacted, and potentially regulated, by dosage compensation. Therefore, I'm investigating the localization of basal transcription machinery within C. elegans genome to define the sites of initiation and the promoters that guide initiation. The changes in the levels of specific GTFs on dosage compensated promoters between wild type and dosage compensation mutants will identify points in PIC assembly altered by dosage compensation to decrease X chromosome gene expression in hermaphrodites.

Severson, Aaron et al. (2009) International Worm Meeting "The axial element protein HTP-3 promotes cohesin loading and assembly of the meiotic chromosomal axis."

Meyer, Barbara, Severson, Aaron

[International Worm Meeting, 2009]

Faithful transmission of the genome through sexual reproduction requires precise reduction of genome copy number during gametogenesis to produce haploid sperm and eggs. Meiosis therefore entails unique steps, that are absent from mitosis, to tether homologous chromosomes together during prophase of meiosis I and to separate homologs and then sister chromatids during anaphase of meiosis I and II. We show that HTP-3, a known component of the axial element (AE) that assembles along meiotic chromosomes and promotes crossover recombination, molecularly links these meiotic innovations. When meiosis begins, sister chromatids are held together by sister chromatid cohesion (SCC), mediated by a protein complex called cohesin. Homologs become linked during crossover recombination. Once recombination is complete, SCC around the crossover holds homologs and sisters together. Their successive separation requires the stepwise proteolysis of Rec8, a meiosis-specific cohesin subunit. During meiosis I, cohesin regulators protect Rec8 locally, at discrete domains of each homolog pair, to keep sisters together. We have found that global regulation of cohesin loading by HTP-3 is also required to forestall sister separation in anaphase I, and that cohesin, in turn, is required for HTP-3 loading and AE assembly. Unexpectedly, REC-8, the known REC-8 paralog COH-3 and the previously unknown paralog COH-4 are together essential for AE assembly. In contrast, REC-8 alone can keep sisters together after anaphase I; consequently, sister chromatids segregate away from one another in meiosis I of rec-8 mutants (premature equational division). In a genetic screen for additional factors required to maintain SCC until meiosis II, we identified HTP-3, already known to promote meiotic double strand DNA break formation, homolog pairing, synapsis and recombination. We show that HTP-3 recruits all known AE components to meiotic chromosomes. Additionally, HTP-3 promotes loading of REC-8 containing cohesin complexes, the first demonstrated requirement for an AE protein in cohesin axis assembly. In htp-3 mutants, sister chromatids separate equationally in anaphase I. Thus, HTP-3 is required for multiple events that distinguish meiosis from mitosis. Moreover, our data suggest that interdependent loading of HTP-3 and cohesin is a principal step in assembly of the meiotic chromosomal axis.

Wheeler, Bayly et al. (2011) International Worm Meeting "Linking dosage compensation complex binding to chromosome-wide gene regulation in C. elegans."

Meyer, Barbara, Wheeler, Bayly

[International Worm Meeting, 2011]

Dosage compensation (DC) is an essential process required to balance levels of gene expression between the two X chromosomes of females and the single X of males. In C. elegans, like flies and mammals, DC modulates gene expression across an entire chromosome and thereby serves as an exemplary system to understand long-range mechanisms of gene regulation. To enact DC, the dosage compensation complex (DCC) is recruited to the X chromosome by recruitment elements, called rex sites, that act at a distance to control gene expression. How these recruitment sites function to recruit the DCC, facilitate spreading, and control gene expression are unknown. Progress here requires the ability to identify compensated genes with high confidence and resolution along X and to manipulate rex sites in their endogenous context. Whole transcriptome sequencing of wild-type and DC-defective animals is being used to identify compensated genes at multiple developmental stages with enhanced sensitivity over previous approaches. In addition, two novel technologies are being employed to insert and delete rex sites in the context of the X to link rex sites with their role in DCC binding, chromosome architecture, and DC. Lastly, parameters that influence the interaction between rex sites and compensated genes are being dissected by assessing expression of a reporter moved throughout the X and autosomes in different epigenetic contexts and at different distances from endogenous and engineered rex sites. Identification of the genes that are compensated coupled with the ability to manipulate rex elements and compensated genes will uncover mechanisms by which regulatory elements on the X enact appropriate patterns of gene expression chromosome-wide. In C. elegans and mammals, DC is achieved by decreasing expression from the female X chromosomes to equal the level of expression from the single male X. While this process balances expression between the sexes, it could result in reduced expression from the sex chromosomes relative to the autosomes. It has been proposed that a secondary mechanism of chromosome-wide gene regulation exists to resolve this imbalance by increasing gene expression from the X in both sexes. Support for this hypothesis remains controversial. Therefore, the expression of both endogenous and engineered genes on the X and autosomes is being compared to test the existence of a secondary mechanism of chromosome-wide gene regulation; one that balances X gene expression in both sexes with the autosomes.

Krassovsky, Kristina et al. (2015) International Worm Meeting "Spatial organization of chromosomes in polyploid nuclei of C. elegans."

Meyer, Barbara J, Krassovsky, Kristina

[International Worm Meeting, 2015]

Polyploidy is a cell property that indicates presence of multiple copies of the genome. During polyploidization, the nucleus increases in size, thus decreasing the surface to volume ratio. In the eukaryotic cell, gene transcription occurs in the context of packaged DNA, and different aspects of this packaging and nuclear organization may influence gene transcription. For example, genes positioned close to the nuclear membrane and associated with nuclear lamins are usually silent. Peripherally positioned genes that interact with a nuclear pore complex are active, and this association is often necessary for optimal expression. We are first addressing two key questions. How does chromosome organization change with increase in ploidy? Do polyploid cells equalize DNA packaging factors such as position inside the nucleus, e.g., proximity to lamins and nuclear pores, in order to proportionately transcribe gene copies?Classical work showed that the intestinal nuclei of C. elegans hermaphrodites increase their ploidy from 2 to 32 during development from the L1 stage to adulthood. Unlike in polytene chromosomes of Drosophila, identical gene copies in guts are spatially separated, as shown by FISH studies of ours and others. These observations make the C. elegans intestine a good model for studying gene copy transcription in polyploid cells; however, the exact details of this DNA content amplification and intestinal chromosome structure are unknown. Using fluorescent in situ hybridization (FISH) to visualize individual gene copies, immunofluorescence (IF) to visualize nuclear pores and lamins, and next generation sequencing of hand-dissected intestines we set out to obtain a comprehensive picture of DNA content, chromosome structure and spatial organization of gene copies inside the worm's intestinal nuclei. Because males have a transcription program distinct from that of hermaphrodites, we are performing our studies in both sexes and extending classical ploidy measurements to males. We anticipate that this spatial map of polyploid nuclei can be used to infer the presence or absence of a mechanism that equalizes distances of gene copies relative to nuclear lamins and nuclear pores. This question is of general interest because amplified genomes are common in both healthy organisms and cancer cells.

Severson, Aaron F. et al. (2013) International Worm Meeting "Interchangeable a-kleisin subunits specify meiotic cohesin function in C. elegans."

Meyer, Barbara J., Severson, Aaron F.

[International Worm Meeting, 2013]

Faithful transmission of the genome through sexual reproduction requires reduction of genome copy number during meiosis to produce haploid sperm and eggs. To achieve this, homologous chromosomes become linked through crossover recombination, the two sister chromatids of each homolog attach to microtubules from the same spindle pole (co-orient) in meiosis I and from opposite spindle poles (bi-orient) in meiosis II, and sister chromatid cohesion (SCC) is released in two steps to allow separation of homologs before sisters. In yeast, reducing ploidy during meiosis requires that Scc1, the "kleisin" subunit of cohesin complexes that mediate mitotic SCC, is replaced by the meiosis-specific paralog Rec8. We have shown that REC-8 is not the sole meiotic kleisin in C. elegans, and we predicted that this was also true in plants and mammals. This has now been proven. In C. elegans, REC-8 and two functionally redundant kleisins called COH-3 and COH-4 (henceforth, COH-3/4) perform specialized roles, indicating that interchangeable kleisin subunits determine cohesin function during meiosis. For example, only REC-8 cohesin can co-orient sister chromatids and mediate SCC that persists after anaphase I. Kleisin identity also influences the mechanism by which cohesin loads onto chromosomes and establishes cohesion between sisters. The axial element protein HTP-3 is required for loading of REC-8, but not COH-3/4. Once loaded, COH-3/4 cohesin is triggered to become cohesive by SPO-11-dependent double strand DNA breaks, while REC-8 cohesin generates SCC independently of SPO-11. Finally, REC-8 and COH-3/4 become asymmetrically distributed on meiotic chromosomes late in prophase of meiosis I: COH-3/4 becomes enriched where SCC is released at anaphase I and REC-8 becomes enriched where sister chromatids co-orient and SCC persists until anaphase II. Because REC-8 alone can co-orient sisters and mediate SCC that persists following anaphase I, we are testing whether achieving this reciprocal pattern of cohesin localization facilitates the stepwise separation of homologs and sister chromatids.

Kruesi, William S. et al. (2011) International Worm Meeting "Targeting the dosage compensation complex to X and mechanism of transcriptional regulation by dosage compensation."

Meyer, Barbara J., Kruesi, William S.

[International Worm Meeting, 2011]

The Caenorhabditis elegans dosage compensation complex (DCC) reduces X-linked transcript levels in XX hermaphrodites to equalize X-chromosome gene expression with that of XO males. The DCC binds to two types of site on X: rex sites that autonomously recruit the DCC in a sequence-dependent manner, and dox sites that fail to recruit the DCC when detached from X and lie within the promoters of highly expressed genes. No correlation is apparent between DCC binding in a promoter and the dosage compensation status of that gene. We have examined the cis and trans features that achieve X-specific targeting of the DCC and the mechanism by which the DCC controls transcript levels. We demonstrated that a subunit of C. elegans MLL/COMPASS, a gene-activation complex, acts within the DCC, a condensin complex, to target the DCC to both X chromosomes of hermaphrodites and thereby reduce gene expression. We also showed that sex-specific DCC recruitment to rex sites by XX-specific factors greatly elevates DCC binding to dox sites in cis, which lack intrinsically high DCC affinity on their own, thereby allowing the DCC to disperse along X. Many rex sites contain a Motif Enriched on X (MEX) that is required for recruiting the DCC to individual sites, but many rex sites lack a MEX motif. Therefore, not all features that contribute to X-specific recognition by the DCC have been identified. We performed ChIP-seq to determine the strongest DCC binding sites and to find additional X-specific features essential for DCC bindng. These experiments allowed us to predict additional rex sites and find additional motifs that are enriched on X and function in X-specific recruitment of the DCC. Identification of new motifs revealed a feature of strongly bound sites: the new motifs and MEX often cluster with a constant orientation and spacing. This result suggests that different proteins or domains within the DCC make unique contacts with different DNA sequences, providing clues as to how the DCC assembles on rex sites. We recently determined that dosage compensation acts, in part, by controlling transcription of X-linked genes. These studies failed to determine the primary step of transcription controlled by the DCC. To characterize dosage compensation's effect on transcription, we are mapping the position of transcriptionally engaged RNA Polymerase II in wild type and dosage compensation mutants using Global Run-On Sequencing (GRO-seq). This GRO-seq data will not only allow us to determine the stage of transcription controlled by dosage compensation, but will also provide insights into transcriptional processes unique to C. elegans, such as trans-splicing and operons.

Lee, Teresa W. et al. (2011) International Worm Meeting "Condensin-mediated chromosome architecture & crossover regulation."

Meyer, Barbara J., Lee, Teresa W.

[International Worm Meeting, 2011]

During meiosis, chromosomes undergo complex morphological changes that ensure their proper segregation. Crossover recombination (CO) supplies a physical connection between homologs critical for successful meiosis. Due to their importance, COs are subject to strict control that guarantees at least one per homolog and ensures wide spacing between multiple COs (interference), which requires communication along an entire chromosome's length. C. elegans exhibits an extreme form of interference: only one CO occurs on each homolog. Meiotic disruption of condensin I or condensin II - complexes that structure chromosomes in preparation for cell division - perturbs CO regulation: CO frequencies are increased, and their distribution along the chromosome is altered. This increase is strongly correlated with an overall extension of the chromosome axis, an increase in DNA double-strand breaks (DSBs), and a shift in DSB distribution to the same genetic intervals as the shifted COs. Condensins have non-redundant roles in CO regulation. Disruption of each causes a different distribution of DSBs and COs, and disruption of both perturbs axis length greater than disruption of either. Both complexes may be deployed differentially while sharing an underlying mechanism for structuring meiotic chromosomes. To evaluate the independent roles of condensin I and II, and possible functional redundancy between subunit paralogs, we are analyzing changes in CO and DSB distributions, and effects on chromosome structure, in different backgrounds that disrupt both complexes. We have also found that depletion of the post-translational modification SUMO perturbs regulation of COs and DSBs, while lengthening chromosomal axes. However, the means by which loss of sumoylation increases COs remains unclear. Sumoylation and condensins can influence global chromosome architecture, permitting chromosome-wide communication that may inform CO regulation.