Cooperation between a hierarchical set of recruitment sites targets the X chromosome for dosage compensation.

In many organisms, it remains unclear how X chromosomes are specified for dosage compensation, since DNA sequence motifs shown to be important for dosage compensation complex (DCC) recruitment are themselves not X-specific. Here, we addressed this problem in C. elegans. We found that the DCC recruiter, SDC-2, is required to maintain open chromatin at a small number of primary DCC recruitment sites, whose sequence and genomic context are X-specific. Along the X, primary recruitment sites are interspersed with secondary sites, whose function is X-dependent. A secondary site can ectopically recruit the DCC when additional recruitment sites are inserted either in tandem or at a distance (>30 kb). Deletion of a recruitment site on the X results in reduced DCC binding across several megabases surrounded by topologically associating domain (TAD) boundaries. Our work elucidates that hierarchy and long-distance cooperativity between gene-regulatory elements target a single chromosome for regulation.

Developmental Dynamics of X-Chromosome Dosage Compensation by the DCC and H4K20me1 in C. elegans.

In Caenorhabditis elegans, the dosage compensation complex (DCC) specifically binds to and represses transcription from both X chromosomes in hermaphrodites. The DCC is composed of an X-specific condensin complex that interacts with several proteins. During embryogenesis, DCC starts localizing to the X chromosomes around the 40-cell stage, and is followed by X-enrichment of H4K20me1 between 100-cell to comma stage. Here, we analyzed dosage compensation of the X chromosome between sexes, and the roles of dpy-27 (condensin subunit), dpy-21 (non-condensin DCC member), set-1 (H4K20 monomethylase) and set-4 (H4K20 di-/tri-methylase) in X chromosome repression using mRNA-seq and ChIP-seq analyses across several developmental time points. We found that the DCC starts repressing the X chromosomes by the 40-cell stage, but X-linked transcript levels remain significantly higher in hermaphrodites compared to males through the comma stage of embryogenesis. Dpy-27 and dpy-21 are required for X chromosome repression throughout development, but particularly in early embryos dpy-27 and dpy-21 mutations produced distinct expression changes, suggesting a DCC independent role for dpy-21. We previously hypothesized that the DCC increases H4K20me1 by reducing set-4 activity on the X chromosomes. Accordingly, in the set-4 mutant, H4K20me1 increased more from the autosomes compared to the X, equalizing H4K20me1 level between X and autosomes. H4K20me1 increase on the autosomes led to a slight repression, resulting in a relative effect of X derepression. H4K20me1 depletion in the set-1 mutant showed greater X derepression compared to equalization of H4K20me1 levels between X and autosomes in the set-4 mutant, indicating that H4K20me1 level is important, but X to autosomal balance of H4K20me1 contributes slightly to X-repression. Thus H4K20me1 is not only a downstream effector of the DCC [corrected].In summary, X chromosome dosage compensation starts in early embryos as the DCC localizes to the X, and is strengthened in later embryogenesis by H4K20me1.

Sex-biased gene expression and evolution of the x chromosome in nematodes.

Studies of X chromosome evolution in various organisms have indicated that sex-biased genes are nonrandomly distributed between the X and autosomes. Here, to extend these studies to nematodes, we annotated and analyzed X chromosome gene content in four Caenorhabditis species and in Pristionchus pacificus. Our gene expression analyses comparing young adult male and female mRNA-seq data indicate that, in general, nematode X chromosomes are enriched for genes with high female-biased expression and depleted of genes with high male-biased expression. Genes with low sex-biased expression do not show the same trend of X chromosome enrichment and depletion. Combined with the observation that highly sex-biased genes are primarily expressed in the gonad, differential distribution of sex-biased genes reflects differences in evolutionary pressures linked to tissue-specific regulation of X chromosome transcription. Our data also indicate that X dosage imbalance between males (XO) and females (XX) is influential in shaping both expression and gene content of the X chromosome. Predicted upregulation of the single male X to match autosomal transcription (Ohno's hypothesis) is supported by our observation that overall transcript levels from the X and autosomes are similar for highly expressed genes. However, comparison of differentially located one-to-one orthologs between C. elegans and P. pacificus indicates lower expression of X-linked orthologs, arguing against X upregulation. These contradicting observations may be reconciled if X upregulation is not a global mechanism but instead acts locally on a subset of tissues and X-linked genes that are dosage sensitive.

Genome-wide analysis of condensin binding in Caenorhabditis elegans.

BACKGROUND: Condensins are multi-subunit protein complexes that are essential for chromosome condensation during mitosis and meiosis, and play key roles in transcription regulation during interphase. Metazoans contain two condensins, I and II, which perform different functions and localize to different chromosomal regions. Caenorhabditis elegans contains a third condensin, I(DC), that is targeted to and represses transcription of the X chromosome for dosage compensation. RESULTS: To understand condensin binding and function, we performed ChIP-seq analysis of C. elegans condensins in mixed developmental stage embryos, which contain predominantly interphase nuclei. Condensins bind to a subset of active promoters, tRNA genes and putative enhancers. Expression analysis in kle-2-mutant larvae suggests that the primary effect of condensin II on transcription is repression. A DNA sequence motif, GCGC, is enriched at condensin II binding sites. A sequence extension of this core motif, AGGG, creates the condensin IDC motif. In addition to differences in recruitment that result in X-enrichment of condensin I(DC) and condensin II binding to all chromosomes, we provide evidence for a shared recruitment mechanism, as condensin I(DC) recruiter SDC-2 also recruits condensin II to the condensin I(DC) recruitment sites on the X. In addition, we found that condensin sites overlap extensively with the cohesin loader SCC-2, and that SDC-2 also recruits SCC-2 to the condensin I(DC) recruitment sites. CONCLUSIONS: Our results provide the first genome-wide view of metazoan condensin II binding in interphase, define putative recruitment motifs, and illustrate shared loading mechanisms for condensin I(DC) and condensin II.

Enhancers regulate progression of development in mammalian cells.

During development and differentiation of an organism, accurate gene regulation is central for cells to maintain and balance their differentiation processes. Transcriptional interactions between cis-acting DNA elements such as promoters and enhancers are the basis for precise and balanced transcriptional regulation. We identified modules of combinations of binding sites in proximal and distal regulatory regions upstream of all transcription start sites (TSSs) in silico and applied these modules to gene expression time-series of mouse embryonic development and differentiation of human stem cells. In addition to tissue-specific regulation controlled by combinations of transcription factors (TFs) binding at promoters, we observed that in particular the combination of TFs binding at promoters together with TFs binding at the respective enhancers regulate highly specifically temporal progression during development: whereas 40% of TFs were specific for time intervals, 79% of TF pairs and even 97% of promoter-enhancer modules showed specificity for single time intervals of the human stem cells. Predominantly SP1 and E2F contributed to temporal specificity at promoters and the forkhead (FOX) family of TFs at enhancer regions. Altogether, we characterized three classes of TFs: with binding sites being enriched at the TSS (like SP1), depleted at the TSS (like FOX), and rather uniformly distributed.

PathWave: discovering patterns of differentially regulated enzymes in metabolic pathways.

MOTIVATION: Gene expression profiling by microarrays or transcript sequencing enables observing the pathogenic function of tumors on a mesoscopic level. RESULTS: We investigated neuroblastoma tumors that clinically exhibit a very heterogeneous course ranging from rapid growth with fatal outcome to spontaneous regression and detected regulatory oncogenetic shifts in their metabolic networks. In contrast to common enrichment tests, we took network topology into account by applying adjusted wavelet transforms on an elaborated and new 2D grid representation of curated pathway maps from the Kyoto Enzyclopedia of Genes and Genomes. The aggressive form of the tumors showed regulatory shifts for purine and pyrimidine biosynthesis as well as folate-mediated metabolism of the one-carbon pool in respect to increased nucleotide production. We spotted an oncogentic regulatory switch in glutamate metabolism for which we provided experimental validation, being the first steps towards new possible drug therapy. The pattern recognition method we used complements normal enrichment tests to detect such functionally related regulation patterns. AVAILABILITY AND IMPLEMENTATION: PathWave is implemented in a package for R (www.r-project.org) version 2.6.0 or higher. It is freely available from http://www.ichip.de/software/pathwave.html.