Per Stenberg

Alternative Epigenetic Chromatin States of Polycomb Target Genes

Polycomb (PcG) regulation has been thought to produce stable long-term gene silencing. Genomic analyses in Drosophila and mammals, however, have shown that it targets many genes, which can switch state during development. Genetic evidence indicates that critical for the active state of PcG target genes are the histone methyltransferases Trithorax (TRX) and ASH1. Here we analyze the repertoire of alternative states in which PcG target genes are found in different Drosophila cell lines and the role of PcG proteins TRX and ASH1 in controlling these states. Using extensive genome-wide chromatin immunoprecipitation analysis, RNAi knockdowns, and quantitative RT–PCR, we show that, in addition to the known repressed state, PcG targets can reside in a transcriptionally active state characterized by formation of an extended domain enriched in ASH1, the N-terminal, but not C-terminal moiety of TRX and H3K27ac. ASH1/TRX N-ter domains and transcription are not incompatible with repressive marks, sometimes resulting in a “balanced” state modulated by both repressors and activators. Often however, loss of PcG repression results instead in a “void” state, lacking transcription, H3K27ac, or binding of TRX or ASH1. We conclude that PcG repression is dynamic, not static, and that the propensity of a target gene to switch states depends on relative levels of PcG, TRX, and activators. N-ter TRX plays a remarkable role that antagonizes PcG repression and preempts H3K27 methylation by acetylation. This role is distinct from that usually attributed to TRX/MLL proteins at the promoter. These results have important implications for Polycomb gene regulation, the “bivalent” chromatin state of embryonic stem cells, and gene expression in development.

Buffering of Segmental and Chromosomal Aneuploidies in Drosophila melanogaster

Chromosomal instability, which involves the deletion and duplication of chromosomes or chromosome parts, is a common feature of cancers, and deficiency screens are commonly used to detect genes involved in various biological pathways. However, despite their importance, the effects of deficiencies, duplications, and chromosome losses on the regulation of whole chromosomes and large chromosome domains are largely unknown. Therefore, to explore these effects, we examined expression patterns of genes in several Drosophila deficiency hemizygotes and a duplication hemizygote using microarrays. The results indicate that genes expressed in deficiency hemizygotes are significantly buffered, and that the buffering effect is general rather than being mainly mediated by feedback regulation of individual genes. In addition, differentially expressed genes in haploid condition appear to be generally more strongly buffered than ubiquitously expressed genes in haploid condition, but, among genes present in triploid condition, ubiquitously expressed genes are generally more strongly buffered than differentially expressed genes. Furthermore, we show that the 4th chromosome is compensated in response to dose differences. Our results suggest general mechanisms have evolved that stimulate or repress gene expression of aneuploid regions as appropriate, and on the 4th chromosome of Drosophila this compensation is mediated by Painting of Fourth (POF).

Painting of fourth and chromosome‐wide regulation of the 4th chromosome in Drosophila melanogaster

Drosophila melanogaster exhibits two expression‐regulating systems that target whole, specific chromosomes: the dosage compensation system whereby the male‐specific lethal complex doubles transcription of genes on the male X‐chromosome and the chromosome 4‐specific protein Painting of fourth, POF. POF is the first example of an autosome‐specific protein and its presence raises the question of the universality of chromosome‐specific regulation. Here we show that POF and heterochromatin protein 1 (HP1) are involved in the global regulation of the 4th chromosome. Contrary to previous conclusions, Pof is not essential for survival of diplo‐4th karyotype flies. However, Pof is essential for survival of haplo‐4th individuals and expression of chromosome 4 genes in diplo‐4th individuals is decreased in the absence of Pof. Mapping of POF using chromatin immunoprecipitation suggested that it binds within genes. Furthermore, we show that POF binding is dependent on heterochromatin and that POF and HP1 bind interdependently to the 4th chromosome. We propose a balancing mechanism involving POF and HP1 that provides a feedback system for fine‐tuning expression status of genes on the 4th chromosome.

Scaffolding of a bacterial genome using MinION nanopore sequencing

Second generation sequencing has revolutionized genomic studies. However, most genomes contain repeated DNA elements that are longer than the read lengths achievable with typical sequencers, so the genomic order of several generated contigs cannot be easily resolved. A new generation of sequencers offering substantially longer reads is emerging, notably the Pacific Biosciences (PacBio) RS II system and the MinION system, released in early 2014 by Oxford Nanopore Technologies through an early access program. The latter has highly advantageous portability and sequences samples by measuring changes in ionic current when single-stranded DNA molecules are translocated through nanopores. We show that the MinION system produces long reads with high mapability that can be used for scaffolding bacterial genomes, despite currently producing substantially higher error rates than PacBio reads. With further development we anticipate that MinION will be useful not only for assembling genomes, but also for rapid detection of organisms, potentially in the field.

Buffering and the evolution of chromosome-wide gene regulation

Copy number variation (CNV) in terms of aneuploidies of both entire chromosomes and chromosomal segments is an important evolutionary driving force, but it is inevitably accompanied by potentially problematic variations in gene doses and genomic instability. Thus, a delicate balance must be maintained between mechanisms that compensate for variations in gene doses (and thus allow such genomic variability) and selection against destabilizing CNVs. In Drosophila, three known compensatory mechanisms have evolved: a general segmental aneuploidy-buffering system and two chromosome-specific systems. The two chromosome-specific systems are the male-specific lethal complex, which is important for dosage compensation of the male X chromosome, and Painting of fourth, which stimulates expression of the fourth chromosome. In this review, we discuss the origin and function of buffering and compensation using Drosophila as a model.

Cytology of Asexual Animals

We review the cytological mechanisms underlying asexual reproduction, i.e. reproduction without fertilization, in animals. Asexuality or parthenogenesis has evolved many times and the cytological mechanisms to restore the parental chromosome number can vary between and even within species. In automictic or meiotic parthenogenesis, meiosis takes place but the chromosomal constitution of the mother is restored through one or several different mechanisms. Some of these mechanisms enforce homozygosity at all loci while some other mechanisms pass the genome of the mother intact to the offspring. In apomictic or mitotic parthenogenesis the eggs are formed through what is essentially a set of mitoses. Polyploidy, is in general incompatible with chromosomal sex determination and is a rare condition in animals. However, many asexual and hermaphroditic forms are polyploid to various degrees. Polyploidy is divided into allo- and autopolyploidy. In the former mode the chromosome sets are derived from two or more different species while in autopolyploidy the multiplication has taken place within one species. We discuss the evolutionary consequences of the different cytological mechanisms involved in asexual reproduction.

Distribution, mechanisms and evolutionary significance of clonality and polyploidy in weevils

Abstract  1 Genetical mtDNA relationships of 41 taxa of weevils were examined using cladistics. Ingroup taxa belong to Otiorhynchus scaber and O. nodosus and outgroup comparison was made with O. singularis. All three species are minor forest pests.

Combinatorial Interactions Are Required for the Efficient Recruitment of Pho Repressive Complex (PhoRC) to Polycomb Response Elements

Polycomb Group (PcG) proteins are epigenetic repressors that control metazoan development and cell differentiation. In Drosophila, PcG proteins form five distinct complexes targeted to genes by Polycomb Response Elements (PREs). Of all PcG complexes PhoRC is the only one that contains a sequence-specific DNA binding subunit (PHO or PHOL), which led to a model that places PhoRC at the base of the recruitment hierarchy. Here we demonstrate that in vivo PHO is preferred to PHOL as a subunit of PhoRC and that PHO and PHOL associate with PREs and a subset of transcriptionally active promoters. Although the binding to the promoter sites depends on the quality of recognition sequences, the binding to PREs does not. Instead, the efficient recruitment of PhoRC to PREs requires the SFMBT subunit and crosstalk with Polycomb Repressive Complex 1. We find that human YY1 protein, the ortholog of PHO, binds sites at active promoters in the human genome but does not bind most PcG target genes, presumably because the interactions involved in the targeting to Drosophila PREs are lost in the mammalian lineage. We conclude that the recruitment of PhoRC to PREs is based on combinatorial interactions and propose that such a recruitment strategy is important to attenuate the binding of PcG proteins when the target genes are transcriptionally active. Our findings allow the appropriate placement of PhoRC in the PcG recruitment hierarchy and provide a rationale to explain why YY1 is unlikely to serve as a general recruiter of mammalian Polycomb complexes despite its reported ability to participate in PcG repression in flies.

HP1a Recruitment to Promoters Is Independent of H3K9 Methylation in Drosophila melanogaster

Heterochromatin protein 1 (HP1) proteins, recognized readers of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me), are important regulators of heterochromatin-mediated gene silencing and chromosome structure. In Drosophila melanogaster three histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9: Su(var)3-9, Setdb1, and G9a. To probe the dependence of HP1a binding on H3K9me, its dependence on these three HKMTs, and the division of labor between the HKMTs, we have examined correlations between HP1a binding and H3K9me patterns in wild type and null mutants of these HKMTs. We show here that Su(var)3-9 controls H3K9me-dependent binding of HP1a in pericentromeric regions, while Setdb1 controls it in cytological region 2L:31 and (together with POF) in chromosome 4. HP1a binds to the promoters and within bodies of active genes in these three regions. More importantly, however, HP1a binding at promoters of active genes is independent of H3K9me and POF. Rather, it is associated with heterochromatin protein 2 (HP2) and open chromatin. Our results support a hypothesis in which HP1a nucleates with high affinity independently of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites.

Preferential Genome Targeting of the CBP Co-Activator by Rel and Smad Proteins in Early Drosophila melanogaster Embryos

CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with multiple transcription factors. Whether CBP/p300 occupies the genome equally with all factors or preferentially binds together with some factors is not known. We therefore compared Drosophila melanogaster CBP (nejire) ChIP–seq peaks with regions bound by 40 different transcription factors in early embryos, and we found high co-occupancy with the Rel-family protein Dorsal. Dorsal is required for CBP occupancy in the embryo, but only at regions where few other factors are present. CBP peaks in mutant embryos lacking nuclear Dorsal are best correlated with TGF-ß/Dpp-signaling and Smad-protein binding. Differences in CBP occupancy in mutant embryos reflect gene expression changes genome-wide, but CBP also occupies some non-expressed genes. The presence of CBP at silent genes does not result in histone acetylation. We find that Polycomb-repressed H3K27me3 chromatin does not preclude CBP binding, but restricts histone acetylation at CBP-bound genomic sites. We conclude that CBP occupancy in Drosophila embryos preferentially overlaps factors controlling dorso-ventral patterning and that CBP binds silent genes without causing histone hyperacetylation.