Getting started: altering promoter choice as a mechanism for cell type differentiation.

In this issue of Genes & Development, Lu and colleagues (pp. 663-677) have discovered a key new mechanism of alternative promoter choice that is involved in differentiation of spermatocytes. Promoter choice has strong potential as mechanism for differentiation of many different cell types.

Histone locus regulation by the Drosophila dosage compensation adaptor protein CLAMP.

The conserved histone locus body (HLB) assembles prior to zygotic gene activation early during development and concentrates factors into a nuclear domain of coordinated histone gene regulation. Although HLBs form specifically at replication-dependent histone loci, the cis and trans factors that target HLB components to histone genes remained unknown. Here we report that conserved GA repeat cis elements within the bidirectional histone3-histone4 promoter direct HLB formation in Drosophila In addition, the CLAMP (chromatin-linked adaptor for male-specific lethal [MSL] proteins) zinc finger protein binds these GA repeat motifs, increases chromatin accessibility, enhances histone gene transcription, and promotes HLB formation. We demonstrated previously that CLAMP also promotes the formation of another domain of coordinated gene regulation: the dosage-compensated male X chromosome. Therefore, CLAMP binding to GA repeat motifs promotes the formation of two distinct domains of coordinated gene activation located at different places in the genome.

A sequence motif within chromatin entry sites directs MSL establishment on the Drosophila X chromosome.

The Drosophila MSL complex associates with active genes specifically on the male X chromosome to acetylate histone H4 at lysine 16 and increase expression approximately 2-fold. To date, no DNA sequence has been discovered to explain the specificity of MSL binding. We hypothesized that sequence-specific targeting occurs at "chromatin entry sites," but the majority of sites are sequence independent. Here we characterize 150 potential entry sites by ChIP-chip and ChIP-seq and discover a GA-rich MSL recognition element (MRE). The motif is only slightly enriched on the X chromosome ( approximately 2-fold), but this is doubled when considering its preferential location within or 3' to active genes (>4-fold enrichment). When inserted on an autosome, a newly identified site can direct local MSL spreading to flanking active genes. These results provide strong evidence for both sequence-dependent and -independent steps in MSL targeting of dosage compensation to the male X chromosome.

TIMEOR: a web-based tool to uncover temporal regulatory mechanisms from multi-omics data.

Uncovering how transcription factors regulate their targets at DNA, RNA and protein levels over time is critical to define gene regulatory networks (GRNs) and assign mechanisms in normal and diseased states. RNA-seq is a standard method measuring gene regulation using an established set of analysis stages. However, none of the currently available pipeline methods for interpreting ordered genomic data (in time or space) use time-series models to assign cause and effect relationships within GRNs, are adaptive to diverse experimental designs, or enable user interpretation through a web-based platform. Furthermore, methods integrating ordered RNA-seq data with protein-DNA binding data to distinguish direct from indirect interactions are urgently needed. We present TIMEOR (Trajectory Inference and Mechanism Exploration with Omics data in R), the first web-based and adaptive time-series multi-omics pipeline method which infers the relationship between gene regulatory events across time. TIMEOR addresses the critical need for methods to determine causal regulatory mechanism networks by leveraging time-series RNA-seq, motif analysis, protein-DNA binding data, and protein-protein interaction networks. TIMEOR's user-catered approach helps non-coders generate new hypotheses and validate known mechanisms. We used TIMEOR to identify a novel link between insulin stimulation and the circadian rhythm cycle. TIMEOR is available at https://github.com/ashleymaeconard/TIMEOR.git and http://timeor.brown.edu.

Diverse Genome Topologies Characterize Dosage Compensation across Species.

Dosage compensation is the process by which transcript levels of the X chromosome are equalized with those of autosomes. Although diverse mechanisms of dosage compensation have evolved across species, these mechanisms all involve distinguishing the X chromosome from autosomes. Because one chromosome is singled out from other chromosomes for precise regulation, dosage compensation serves as an important model for understanding how specific cis-elements are identified within the highly compacted 3D genome to co-regulate thousands of genes. Recently, multiple genomic approaches have provided key insights into the mechanisms of dosage compensation, extending what we have learned from classical genetic studies. In the future, newer genomic approaches that require little starting material show great promise to provide an understanding of the heterogeneity of dosage compensation between cells and how it functions in nonmodel organisms.

The simultaneous interaction of MSL2 with CLAMP and DNA provides redundancy in the initiation of dosage compensation in Drosophila males.

The binding of the Drosophila male-specific lethal dosage compensation complex (DCC) exclusively to the male X chromosome provides an excellent model system to understand mechanisms of selective recruitment of protein complexes to chromatin. Previous studies showed that the male-specific organizer of the complex, MSL2, and the ubiquitous DNA-binding protein CLAMP are key players in the specificity of X chromosome binding. The CXC domain of MSL2 binds to genomic sites of DCC recruitment in vitro Another conserved domain of MSL2, named Clamp-binding domain (CBD) directly interacts with the N-terminal zinc-finger domain of CLAMP. Here, we found that inactivation of CBD or CXC individually only modestly affected recruitment of the DCC to the X chromosome in males. However, combination of these two genetic lesions within the same MSL2 mutant resulted in an increased loss of DCC recruitment to the X chromosome. Thus, proper MSL2 positioning requires an interaction with either CLAMP or DNA to initiate dosage compensation in Drosophila males.

The CLAMP protein links the MSL complex to the X chromosome during Drosophila dosage compensation.

The Drosophila male-specific lethal (MSL) dosage compensation complex increases transcript levels on the single male X chromosome to equal the transcript levels in XX females. However, it is not known how the MSL complex is linked to its DNA recognition elements, the critical first step in dosage compensation. Here, we demonstrate that a previously uncharacterized zinc finger protein, CLAMP (chromatin-linked adaptor for MSL proteins), functions as the first link between the MSL complex and the X chromosome. CLAMP directly binds to the MSL complex DNA recognition elements and is required for the recruitment of the MSL complex. The discovery of CLAMP identifies a key factor required for the chromosome-specific targeting of dosage compensation, providing new insights into how subnuclear domains of coordinate gene regulation are formed within metazoan genomes.

Comparative analysis of metazoan chromatin organization.

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal 'arms', and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.

Expansion of GA Dinucleotide Repeats Increases the Density of CLAMP Binding Sites on the X-Chromosome to Promote Drosophila Dosage Compensation.

Dosage compensation is an essential process that equalizes transcript levels of X-linked genes between sexes by forming a domain of coordinated gene expression. Throughout the evolution of Diptera, many different X-chromosomes acquired the ability to be dosage compensated. Once each newly evolved X-chromosome is targeted for dosage compensation in XY males, its active genes are upregulated two-fold to equalize gene expression with XX females. In Drosophila melanogaster, the CLAMP zinc finger protein links the dosage compensation complex to the X-chromosome. However, the mechanism for X-chromosome identification has remained unknown. Here, we combine biochemical, genomic and evolutionary approaches to reveal that expansion of GA-dinucleotide repeats likely accumulated on the X-chromosome over evolutionary time to increase the density of CLAMP binding sites, thereby driving the evolution of dosage compensation. Overall, we present new insight into how subtle changes in genomic architecture, such as expansions of a simple sequence repeat, promote the evolution of coordinated gene expression.

The essential Drosophila CLAMP protein differentially regulates non-coding roX RNAs in male and females.

Heterogametic species require chromosome-wide gene regulation to compensate for differences in sex chromosome gene dosage. In Drosophila melanogaster, transcriptional output from the single male X-chromosome is equalized to that of XX females by recruitment of the male-specific lethal (MSL) complex, which increases transcript levels of active genes 2-fold. The MSL complex contains several protein components and two non-coding RNA on the X ( roX) RNAs that are transcriptionally activated by the MSL complex. We previously discovered that targeting of the MSL complex to the X-chromosome is dependent on the chromatin-linked adapter for MSL proteins (CLAMP) zinc finger protein. To better understand CLAMP function, we used the CRISPR/Cas9 genome editing system to generate a frameshift mutation in the clamp gene that eliminates expression of the CLAMP protein. We found that clamp null females die at the third instar larval stage, while almost all clamp null males die at earlier developmental stages. Moreover, we found that in clamp null females roX gene expression is activated, whereas in clamp null males roX gene expression is reduced. Therefore, CLAMP regulates roX abundance in a sex-specific manner. Our results provide new insights into sex-specific gene regulation by an essential transcription factor.

Wisdom from the fly.

Arguably, almost all research in Drosophila can be considered basic research, yet many of the most essential and fundamental concepts of human genetics were first decoded in the fly. Although the fly genome, which is organized into only four chromosomes, is approximately one-twentieth the size of the human genome, it contains roughly the same number of genes, and up to 75% of human disease-related genes have Drosophila homologues [1]. The fly was prized for its simplicity and utility even before such compelling homology with humans was apparent. Since Thomas Hunt Morgan began his seminal experiments over a century ago (Table 1), the Drosophila system has revealed countless key mechanisms by which cells function, including the factors that maintain chromatin and the signaling pathways that control cell fate determination and organism development. More recently, the fly has emerged as a critical neurobiological tool and disease model for a range of genetic disorders. In this review, we present a brief retrospective of Drosophila as an indispensable genetic system and discuss some of the many contributions, past and present, of this facile system to human genetics.

X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila.

The evolution of sex chromosomes has resulted in numerous species in which females inherit two X chromosomes but males have a single X, thus requiring dosage compensation. MSL (Male-specific lethal) complex increases transcription on the single X chromosome of Drosophila males to equalize expression of X-linked genes between the sexes. The biochemical mechanisms used for dosage compensation must function over a wide dynamic range of transcription levels and differential expression patterns. It has been proposed that the MSL complex regulates transcriptional elongation to control dosage compensation, a model subsequently supported by mapping of the MSL complex and MSL-dependent histone 4 lysine 16 acetylation to the bodies of X-linked genes in males, with a bias towards 3' ends. However, experimental analysis of MSL function at the mechanistic level has been challenging owing to the small magnitude of the chromosome-wide effect and the lack of an in vitro system for biochemical analysis. Here we use global run-on sequencing (GRO-seq) to examine the specific effect of the MSL complex on RNA Polymerase II (RNAP II) on a genome-wide level. Results indicate that the MSL complex enhances transcription by facilitating the progression of RNAP II across the bodies of active X-linked genes. Improving transcriptional output downstream of typical gene-specific controls may explain how dosage compensation can be imposed on the diverse set of genes along an entire chromosome.

Comprehensive analysis of the chromatin landscape in Drosophila melanogaster.

Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function.

X-marks the spot: X-chromosome identification during dosage compensation.

Dosage compensation is the essential process that equalizes the dosage of X-linked genes between the sexes in heterogametic species. Because all of the genes along the length of a single chromosome are co-regulated, dosage compensation serves as a model system for understanding how domains of coordinate gene regulation are established. Dosage compensation has been best studied in mammals, flies and worms. Although dosage compensation systems are seemingly diverse across species, there are key shared principles of nucleation and spreading that are critical for accurate targeting of the dosage compensation complex to the X-chromosome(s). We will highlight the mechanisms by which long non-coding RNAs function together with DNA sequence elements to tether dosage compensation complexes to the X-chromosome. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.

A new player in X identification: the CLAMP protein is a key factor in Drosophila dosage compensation.

Dosage compensation adjusts the expression levels of genes on one or both targeted sex chromosomes in heterogametic species. This process results in the normalized transcriptional output of important and essential gene families encoded on multiple chromosomes. The mechanisms of dosage compensation have been studied in many model organisms, including Drosophila melanogaster (fly), Caenorhabditis elegans (worm), and Mus musculus (mouse). Although the mechanisms of dosage compensations differ among these species, all of these processes rely on the initial discrimination of the X chromosome from autosomes. Recently, a new paradigm for how the X chromosome is targeted for regulation was identified in Drosophila. This mechanism involves a newly identified zinc finger protein, CLAMP. Here, we review important factors involved in dosage compensation across species with special focus on the fly. Understanding how the newly identified CLAMP protein is involved in X targeting in the fly could provide key insights into how the X chromosome is initially identified across species.

Identification of chromatin-associated regulators of MSL complex targeting in Drosophila dosage compensation.

Sex chromosome dosage compensation in Drosophila provides a model for understanding how chromatin organization can modulate coordinate gene regulation. Male Drosophila increase the transcript levels of genes on the single male X approximately two-fold to equal the gene expression in females, which have two X-chromosomes. Dosage compensation is mediated by the Male-Specific Lethal (MSL) histone acetyltransferase complex. Five core components of the MSL complex were identified by genetic screens for genes that are specifically required for male viability and are dispensable for females. However, because dosage compensation must interface with the general transcriptional machinery, it is likely that identifying additional regulators that are not strictly male-specific will be key to understanding the process at a mechanistic level. Such regulators would not have been recovered from previous male-specific lethal screening strategies. Therefore, we have performed a cell culture-based, genome-wide RNAi screen to search for factors required for MSL targeting or function. Here we focus on the discovery of proteins that function to promote MSL complex recruitment to "chromatin entry sites," which are proposed to be the initial sites of MSL targeting. We find that components of the NSL (Non-specific lethal) complex, and a previously unstudied zinc-finger protein, facilitate MSL targeting and display a striking enrichment at MSL entry sites. Identification of these factors provides new insight into how MSL complex establishes the specialized hyperactive chromatin required for dosage compensation in Drosophila.

Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context.

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at "entry sites" that contain a consensus sequence motif ("MSL recognition element" or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.

Dual DNA/RNA-binding factor regulates dynamics of hnRNP splicing condensates.

Despite decades of research, mechanisms by which co-transcriptional alternative splicing events are targeted to the correct genomic locations to drive cell fate decisions remain unknown. By combining structural and molecular approaches, we define a new mechanism by which an essential transcription factor (TF) targets co-transcriptional splicing through physical and functional interaction with RNA and RNA binding proteins (RBPs). We show that an essential TF co-transcriptionally regulates sex-specific alternative splicing by directly interacting with a subset of target RNAs on chromatin and modulating the dynamics of hnRNPA2 homolog nuclear splicing condensates.

The CLAMP GA-binding transcription factor regulates heat stress-induced transcriptional repression by associating with 3D loop anchors.

In order to survive when exposed to heat stress (HS), organisms activate stress response genes and repress constitutive gene expression to prevent the accumulation of potentially toxic RNA and protein products. Although many studies have elucidated the mechanisms that drive HS-induced activation of stress response genes across species, little is known about repression mechanisms or how genes are targeted for activation versus repression context-specifically. The mechanisms of heat stress-regulated activation have been well-studied in Drosophila, in which the GA-binding transcription factor GAF is important for activating genes upon heat stress. Here, we show that a functionally distinct GA-binding transcription factor (TF) protein, CLAMP (Chromatin-linked adaptor for MSL complex proteins), is essential for repressing constitutive genes upon heat stress but not activation of the canonical heat stress pathway. HS induces loss of CLAMP-associated 3D chromatin loop anchors associated with different combinations of GA-binding TFs prior to HS if a gene becomes repressed versus activated. Overall, we demonstrate that CLAMP promotes repression of constitutive genes upon HS, and repression and activation are associated with the loss of CLAMP-associated 3D chromatin loops bound by different combinations of GA-binding TFs.

Sex-specific splicing occurs genome-wide during early Drosophila embryogenesis.

Sex-specific splicing is an essential process that regulates sex determination and drives sexual dimorphism. Yet, how early in development widespread sex-specific transcript diversity occurs was unknown because it had yet to be studied at the genome-wide level. We use the powerful Drosophila model to show that widespread sex-specific transcript diversity occurs early in development, concurrent with zygotic genome activation. We also present a new pipeline called time2Splice to quantify changes in alternative splicing over time. Furthermore, we determine that one of the consequences of losing an essential maternally deposited pioneer factor called CLAMP (chromatin-linked adapter for MSL proteins) is altered sex-specific splicing of genes involved in diverse biological processes that drive development. Overall, we show that sex-specific differences in transcript diversity exist even at the earliest stages of development..