Thomas Conrad

Dosage compensation in Drosophila melanogaster: epigenetic fine-tuning of chromosome-wide transcription.

Dosage compensation is an epigenetic mechanism that normalizes gene expression from unequal copy numbers of sex chromosomes. Different organisms have evolved alternative molecular solutions to this task. In Drosophila melanogaster, transcription of the single male X chromosome is upregulated by twofold in a process orchestrated by the dosage compensation complex. Despite this conceptual simplicity, dosage compensation involves multiple coordinated steps to recognize and activate the entire X chromosome. We are only beginning to understand the intriguing interplay between multiple levels of local and long-range chromatin regulation required for the fine-tuned transcriptional activation of a heterogeneous gene population. This Review highlights the known facts and open questions of dosage compensation in D. melanogaster.

The Nonspecific Lethal Complex Is a Transcriptional Regulator in Drosophila

Here, we report the biochemical characterization of the nonspecific lethal (NSL) complex (NSL1, NSL2, NSL3, MCRS2, MBD-R2, and WDS) that associates with the histone acetyltransferase MOF in both Drosophila and mammals. Chromatin immunoprecipitation-Seq analysis revealed association of NSL1 and MCRS2 with the promoter regions of more than 4000 target genes, 70% of these being actively transcribed. This binding is functional, as depletion of MCRS2, MBD-R2, and NSL3 severely affects gene expression genome wide. The NSL complex members bind to their target promoters independently of MOF. However, depletion of MCRS2 affects MOF recruitment to promoters. NSL complex stability is interdependent and relies mainly on the presence of NSL1 and MCRS2. Tethering of NSL3 to a heterologous promoter leads to robust transcription activation and is sensitive to the levels of NSL1, MCRS2, and MOF. Taken together, we conclude that the NSL complex acts as a major transcriptional regulator in Drosophila.

Transcription-Coupled Methylation of Histone H3 at Lysine 36 Regulates Dosage Compensation by Enhancing Recruitment of the MSL Complex in Drosophila melanogaster

ABSTRACT In Drosophila melanogaster, dosage compensation relies on the targeting of the male-specific lethal (MSL) complex to hundreds of sites along the male X chromosome. Transcription-coupled methylation of histone H3 lysine 36 is enriched toward the 3′ end of active genes, similar to the MSL proteins. Here, we have studied the link between histone H3 methylation and MSL complex targeting using RNA interference and chromatin immunoprecipitation. We show that trimethylation of histone H3 at lysine 36 (H3K36me3) relies on the histone methyltransferase Hypb and is localized promoter distal at dosage-compensated genes, similar to active genes on autosomes. However, H3K36me3 has an X-specific function, as reduction specifically decreases acetylation of histone H4 lysine 16 on the male X chromosome. This hypoacetylation is caused by compromised MSL binding and results in a failure to increase expression twofold. Thus, H3K36me3 marks the body of all active genes yet is utilized in a chromosome-specific manner to enhance histone acetylation at sites of dosage compensation.

Drosophila Dosage Compensation Involves Enhanced Pol II Recruitment to Male X-Linked Promoters

Promoting the Male X Chromosome In mammals and fruit flies, females have a double dose of the X chromosome compared to males, and to compensate for this imbalance, in fruit flies, transcription from across most of the male X chromosome is boosted by twofold. Conrad et al. (p. 742, published online 19 July) measured the binding of RNA polymerase II, responsible for the majority of the transcription on the X chromosome, and found a consistent increase at the promoters of genes on the male X chromosome. Thus, the increase in transcription on the male X chromosome is not driven by increased rates of transcriptional elongation, as has been suggested previously, but must involve up-regulation of transcription initiation. Boosting gene expression from the entire X chromosome in males happens mainly at the level of transcription initiation. Through hyperacetylation of histone H4 lysine 16 (H4K16), the male-specific lethal (MSL) complex in Drosophila approximately doubles transcription from the single male X chromosome in order to match X-linked expression in females and expression from diploid autosomes. By obtaining accurate measurements of RNA polymerase II (Pol II) occupancies and short promoter-proximal RNA production, we detected a consistent, genome-scale increase in Pol II activity at the promoters of male X-linked genes. Moreover, we found that enhanced Pol II recruitment to male X-linked promoters is largely dependent on the MSL complex. These observations provide insights into how global modulation of chromatin structure by histone acetylation contributes to the precise control of Pol II function.

The MOF Chromobarrel Domain Controls Genome-wide H4K16 Acetylation and Spreading of the MSL Complex

The histone H4 lysine 16 (H4K16)-specific acetyltransferase MOF is part of two distinct complexes involved in X chromosome dosage compensation and autosomal transcription regulation. Here we show that the MOF chromobarrel domain is essential for H4K16 acetylation throughout the Drosophila genome and is required for spreading of the male-specific lethal (MSL) complex on the X chromosome. The MOF chromobarrel domain directly interacts with nucleic acids and potentiates MOFs enzymatic activity after chromatin binding, making it a unique example of a chromo-like domain directly controlling acetylation activity in vivo. We also show that the Drosophila-specific N terminus of MOF has evolved to perform sex-specific functions. It modulates nucleosome binding and HAT activity and controls MSL complex assembly, thus regulating MOF function in dosage compensation. We propose that MOF has been especially tailored to achieve tight regulation of its enzymatic activity and enable its dual role on X and autosomes.

Response to Comments on "Drosophila Dosage Compensation Involves Enhanced Pol II Recruitment to Male X-Linked Promoters".

Ferrari et al. and Straub and Becker wrongly claim that an error in the computational analysis calls into question the conclusions of Conrad et al. All the available evidence, including the reanalyzed genomic data, show that the conclusions and the key message of the study remain unchanged: RNA polymerase II recruitment to male X-linked promoters is an important regulatory step during dosage compensation.

Cellular Fractionation and Isolation of Chromatin-Associated RNA.

In eukaryotic cells, the synthesis, processing, and functions of RNA molecules are confined to distinct subcellular compartments. Biochemical fractionation of cells prior to RNA isolation thus enables the analysis of distinct steps in the lifetime of individual RNA molecules that would be masked in bulk RNA preparations from whole cells. Here, we describe a simple two-step differential centrifugation protocol for the isolation of cytoplasmic, nucleoplasmic, and chromatin-associated RNA that can be used in downstream applications such as qPCR or deep sequencing. We discuss various aspects of this fractionation protocol, which can be readily applied to many mammalian cell types. For the study of long noncoding RNAs and enhancer RNAs in regulation of transcription especially the preparation of chromatin-associated RNA can contribute significantly to further developments.

Insight into miRNA biogenesis with RNA sequencing

MicroRNAs (miRNA) are small RNAs that posttranscriptionally regulate gene expression, predominantly by modulating the translation and stability of target messenger RNAs [1]. Over the last 15 years, a tremendous expansion of knowledge has changed the perception of miRNAs from a rare peculiarity in nematode worms to a ubiquitous layer of gene expression regulation, with broad roles in development, and cellular and organismic homeostasis across animal and plant species. Accordingly, aberrant miRNA expression is a hallmark of various severe disease phenotypes such as cardiomyopathies or cancer. Nevertheless, despite extensive efforts to dissect miRNA functions, many open questions are still remaining. Especially the upstream events that regulate processing have recently received increasing attention, as the DNA elements that control miRNA expression and the cellular signaling pathways that fine-tune miRNA processing are still poorly characterized. One aspect of the miRNA biogenesis pathway that remains challenging is to understand why these short RNAs are expressed as long primary transcripts (pri-miRNA) that can be several kilobases in length. While several attempts have been made to predict the transcription start sites (TSS) of pri-miRNAs, their validation remains an experimental challenge due to their nuclear localization and decreased stability compared to mRNAs. pri-miRNAs are processed co-transcriptionally to precursor miRNA hairpins (pre-miRNA) by the Microprocessor complex. pre-miRNAs are then further processed by Dicer into mature miRNAs and incorporated into the RNA-induced silencing complex (RISC) to exert their functions [2]. At the same time, the complex can distinguish pri-miRNA transcripts from unrelated RNA stem-loop structures through unknown mechanisms, likely dependent on sequence elements in the flanking regions. In our recent work we show that the endogenous Microprocessor activity towards individual pri-miRNAs can be determined using RNA sequencing of chromatin-associated RNA and random primed library generation [3]. Using this approach we identify the Microprocessor cleavage signature, a pronounced dip in the sequencing reads of chromatin bound pri-miRNAs reminiscent of the cleaved-out pre-miRNA hairpin. Based on the extent of this signature, we define the MicroProcessing Index (MPI) as a measure for processing efficiency. Genome-wide assessment of the MPIs of all expressed pri-miRNAs shows that processing is one of the major determinants for the expression level of individual mature miRNAs, exceeding the contribution from transcriptional or post-processing regulation. The processing efficiency of specific pri-miRNAs is comparable between cell lines, suggesting a major impact of primary sequence and RNA structure, and to a lesser extent a differential regulation by cell-type specific co-factors. We can derive sequence motifs associated with an increase in processing efficiency, partially confirming previous in vitro approaches [4], particularly the CNNC motif (where N is any base) 3′ of the pre-miRNA that has been shown to be important for processing of C. elegans pri-miRNAs in human (4). This in vitro study suggested that proteins such as the SR protein SRP20 can bind to the regions flanking the pre-miRNAs and facilitate processing [4]. The aspect of pri-miRNA TSS was not further addressed in our paper, but it is evident from the data that the accurate definition of the entire pri-miRNA transcript is feasible for abundant pri-miRNAs, as has also been reported by a recent paper applying a similar approach but with oligod(T) priming in the RNA sequencing library preparation [5]. Large-scale sequencing studies keep providing new insights into the general mechanisms of pri-miRNA transcription and processing as it occurs in a cellular context. Two papers published this year use NET-seq to study global transcription by RNA polymerase(II) [6, 7]. These data have also been suggested to provide insight into pri-miRNA processing events that occur co-transcriptionally. From the data it is evident that transcriptional pausing or intermediate cleavage events occur at pre-miRNA sites at chromatin. While the interpretation of these data is not yet conclusive with respect to miRNA expression, they could reveal possible links between pri-miRNA processing and the transcription process. In summary, large-scale approaches using RNA sequencing are proving to be valuable to uncover some of the missing aspects of pri-miRNA transcription and processing, and more assays like these are expected to add further to our understanding of miRNA biogenesis in the near future.

Transient N-6-Methyladenosine Transcriptome Sequencing Reveals a Regulatory Role of m6A in Splicing Efficiency

Splicing efficiency varies among transcripts, and tight control of splicing kinetics is crucial for coordinated gene expression. N-6-methyladenosine (m6A) is the most abundant RNA modification and is involved in regulation of RNA biogenesis and function. The impact of m6A on regulation of RNA splicing kinetics is unknown. Here, we provide a time-resolved high-resolution assessment of m6A on nascent RNA transcripts and unveil its importance for the control of RNA splicing kinetics. We find that early co-transcriptional m6A deposition near splice junctions promotes fast splicing, while m6A modifications in introns are associated with long, slowly processed introns and alternative splicing events. In conclusion, we show that early m6A deposition specifies the fate of transcripts regarding splicing kinetics and alternative splicing.

Functional interplay between MSL1 and CDK7 controls RNA polymerase II Ser5 phosphorylation

Proper gene expression requires coordinated interplay among transcriptional coactivators, transcription factors and the general transcription machinery. We report here that MSL1, a central component of the dosage compensation complex in Drosophila melanogaster and Drosophila virilis, displays evolutionarily conserved sex-independent binding to promoters. Genetic and biochemical analyses reveal a functional interaction of MSL1 with CDK7, a subunit of the Cdk-activating kinase (CAK) complex of the general transcription factor TFIIH. Importantly, MSL1 depletion leads to decreased phosphorylation of Ser5 of RNA polymerase II. In addition, we demonstrate that MSL1 is a phosphoprotein, and transgenic flies expressing MSL1 phosphomutants show mislocalization of the histone acetyltransferase MOF and histone H4 K16 acetylation, thus ultimately causing male lethality due to a failure of dosage compensation. We propose that, by virtue of its interaction with components of the general transcription machinery, MSL1 exists in different phosphorylation states, thereby modulating transcription in flies.