Philge Philip

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.

Male X-linked genes in Drosophila melanogaster are compensated independently of the Male-Specific Lethal complex

BackgroundIn organisms where the two sexes have unequal numbers of X-chromosomes, the expression of X-linked genes needs to be balanced not only between the two sexes, but also between X and the autosomes. In Drosophila melanogaster, the Male-Specific Lethal (MSL) complex is believed to produce a 2-fold increase in expression of genes on the male X, thus restoring this balance.ResultsHere we show that almost all the genes on the male X are effectively compensated. However, many genes are compensated without any significant recruitment of the MSL-complex. These genes are very weakly, if at all, affected by mutations or RNAi against MSL-complex components. In addition, even the genes that are strongly bound by MSL rely on mechanisms other than the MSL-complex for proper compensation. We find that long, non-ubiquitously expressed genes tend to rely less on the MSL-complex for their compensation and genes that in addition are far from High Affinity Sites tend to not bind the complex at all or very weakly.ConclusionsWe conclude that most of the compensation of X-linked genes is produced by an MSL-independent mechanism. Similar to the case of the MSL-mediated compensation we do not yet know the mechanism behind the MSL-independent compensation that appears to act preferentially on long genes. Even if we observe similarities, it remains to be seen if the mechanism is related to the buffering that is observed in autosomal aneuploidies.

CBP binding outside of promoters and enhancers in Drosophila melanogaster

BackgroundCREB-binding protein (CBP, also known as nejire) is a transcriptional co-activator that is conserved in metazoans. CBP plays an important role in embryonic development and cell differentiation and mutations in CBP can lead to various diseases in humans. In addition, CBP and the related p300 protein have successfully been used to predict enhancers in both humans and flies when they occur with monomethylation of histone H3 on lysine 4 (H3K4me1).ResultsHere, we compare CBP chromatin immunoprecipitation sequencing data from Drosophila S2 cells with modENCODE data and show that CBP is bound at genomic sites with a wide range of functions. As expected, we find that CBP is bound at active promoters and enhancers. In addition, we find that the strongest CBP sites in the genome are found at Polycomb response elements embedded in histone H3 lysine 27 trimethylated (H3K27me3) chromatin, where they correlate with binding of the Pho repressive complex. Interestingly, we find that CBP also binds to most insulators in the genome. At a subset of these, CBP may regulate insulating activity, measured as the ability to prevent repressive H3K27 methylation from spreading into adjacent chromatin.ConclusionsWe conclude that CBP could be involved in a much wider range of functions than has previously been appreciated, including Polycomb repression and insulator activity. In addition, we discuss the possibility that a common role for CBP at all functional elements may be to regulate interactions between distant chromosomal regions and speculate that CBP is controlling higher order chromatin organization.

Non-coding roX RNAs Prevent the Binding of the MSL-complex to Heterochromatic Regions

Long non-coding RNAs contribute to dosage compensation in both mammals and Drosophila by inducing changes in the chromatin structure of the X-chromosome. In Drosophila melanogaster, roX1 and roX2 are long non-coding RNAs that together with proteins form the male-specific lethal (MSL) complex, which coats the entire male X-chromosome and mediates dosage compensation by increasing its transcriptional output. Studies on polytene chromosomes have demonstrated that when both roX1 and roX2 are absent, the MSL-complex becomes less abundant on the male X-chromosome and is relocated to the chromocenter and the 4th chromosome. Here we address the role of roX RNAs in MSL-complex targeting and the evolution of dosage compensation in Drosophila. We performed ChIP-seq experiments which showed that MSL-complex recruitment to high affinity sites (HAS) on the X-chromosome is independent of roX and that the HAS sequence motif is conserved in D. simulans. Additionally, a complete and enzymatically active MSL-complex is recruited to six specific genes on the 4th chromosome. Interestingly, our sequence analysis showed that in the absence of roX RNAs, the MSL-complex has an affinity for regions enriched in Hoppel transposable elements and repeats in general. We hypothesize that roX mutants reveal the ancient targeting of the MSL-complex and propose that the role of roX RNAs is to prevent the binding of the MSL-complex to heterochromatin.

Normalization of High Dimensional Genomics Data Where the Distribution of the Altered Variables Is Skewed

Genome-wide analysis of gene expression or protein binding patterns using different array or sequencing based technologies is now routinely performed to compare different populations, such as treatment and reference groups. It is often necessary to normalize the data obtained to remove technical variation introduced in the course of conducting experimental work, but standard normalization techniques are not capable of eliminating technical bias in cases where the distribution of the truly altered variables is skewed, i.e. when a large fraction of the variables are either positively or negatively affected by the treatment. However, several experiments are likely to generate such skewed distributions, including ChIP-chip experiments for the study of chromatin, gene expression experiments for the study of apoptosis, and SNP-studies of copy number variation in normal and tumour tissues. A preliminary study using spike-in array data established that the capacity of an experiment to identify altered variables and generate unbiased estimates of the fold change decreases as the fraction of altered variables and the skewness increases. We propose the following work-flow for analyzing high-dimensional experiments with regions of altered variables: (1) Pre-process raw data using one of the standard normalization techniques. (2) Investigate if the distribution of the altered variables is skewed. (3) If the distribution is not believed to be skewed, no additional normalization is needed. Otherwise, re-normalize the data using a novel HMM-assisted normalization procedure. (4) Perform downstream analysis. Here, ChIP-chip data and simulated data were used to evaluate the performance of the work-flow. It was found that skewed distributions can be detected by using the novel DSE-test (Detection of Skewed Experiments). Furthermore, applying the HMM-assisted normalization to experiments where the distribution of the truly altered variables is skewed results in considerably higher sensitivity and lower bias than can be attained using standard and invariant normalization methods.

Erratum to: CBP binding outside of promoters and enhancers in Drosophila melanogaster

[This corrects the article DOI: 10.1186/s13072-015-0042-4.].