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..

The continuum of Drosophila embryonic development at single-cell resolution.

Drosophila melanogaster is a powerful, long-standing model for metazoan development and gene regulation. We profiled chromatin accessibility in almost 1 million and gene expression in half a million nuclei from overlapping windows spanning the entirety of embryogenesis. Leveraging developmental asynchronicity within embryo collections, we applied deep neural networks to infer the age of each nucleus, resulting in continuous, multimodal views of molecular and cellular transitions in absolute time. We identify cell lineages; infer their developmental relationships; and link dynamic changes in enhancer usage, transcription factor (TF) expression, and the accessibility of TFs' cognate motifs. With these data, the dynamics of enhancer usage and gene expression can be explored within and across lineages at the scale of minutes, including for precise transitions like zygotic genome activation.

Integrating Long-Range Regulatory Interactions to Predict Gene Expression Using Graph Convolutional Networks.

Long-range regulatory interactions among genomic regions are critical for controlling gene expression, and their disruption has been associated with a host of diseases. However, when modeling the effects of regulatory factors, most deep learning models either neglect long-range interactions or fail to capture the inherent 3D structure of the underlying genomic organization. To address these limitations, we present a Graph Convolutional Model for Epigenetic Regulation of Gene Expression (GC-MERGE). Using a graph-based framework, the model incorporates important information about long-range interactions via a natural encoding of genomic spatial interactions into the graph representation. It integrates measurements of both the global genomic organization and the local regulatory factors, specifically histone modifications, to not only predict the expression of a given gene of interest but also quantify the importance of its regulatory factors. We apply GC-MERGE to data sets for three cell lines-GM12878 (lymphoblastoid), K562 (myelogenous leukemia), and HUVEC (human umbilical vein endothelial)-and demonstrate its state-of-the-art predictive performance. Crucially, we show that our model is interpretable in terms of the observed biological regulatory factors, highlighting both the histone modifications and the interacting genomic regions contributing to a gene's predicted expression. We provide model explanations for multiple exemplar genes and validate them with evidence from the literature. Our model presents a novel setup for predicting gene expression by integrating multimodal data sets in a graph convolutional framework. More importantly, it enables interpretation of the biological mechanisms driving the model's predictions.

Sex-specific aging in animals: Perspective and future directions.

Sex differences in aging occur in many animal species, and they include sex differences in lifespan, in the onset and progression of age-associated decline, and in physiological and molecular markers of aging. Sex differences in aging vary greatly across the animal kingdom. For example, there are species with longer-lived females, species where males live longer, and species lacking sex differences in lifespan. The underlying causes of sex differences in aging remain mostly unknown. Currently, we do not understand the molecular drivers of sex differences in aging, or whether they are related to the accepted hallmarks or pillars of aging or linked to other well-characterized processes. In particular, understanding the role of sex-determination mechanisms and sex differences in aging is relatively understudied. Here, we take a comparative, interdisciplinary approach to explore various hypotheses about how sex differences in aging arise. We discuss genomic, morphological, and environmental differences between the sexes and how these relate to sex differences in aging. Finally, we present some suggestions for future research in this area and provide recommendations for promising experimental designs.

CLAMP and Zelda function together to promote Drosophila zygotic genome activation.

During the essential and conserved process of zygotic genome activation (ZGA), chromatin accessibility must increase to promote transcription. Drosophila is a well-established model for defining mechanisms that drive ZGA. Zelda (ZLD) is a key pioneer transcription factor (TF) that promotes ZGA in the Drosophila embryo. However, many genomic loci that contain GA-rich motifs become accessible during ZGA independent of ZLD. Therefore, we hypothesized that other early TFs that function with ZLD have not yet been identified, especially those that are capable of binding to GA-rich motifs such as chromatin-linked adaptor for male-specific lethal (MSL) proteins (CLAMP). Here, we demonstrate that Drosophila embryonic development requires maternal CLAMP to (1) activate zygotic transcription; (2) increase chromatin accessibility at promoters of specific genes that often encode other essential TFs; and (3) enhance chromatin accessibility and facilitate ZLD occupancy at a subset of key embryonic promoters. Thus, CLAMP functions as a pioneer factor that plays a targeted yet essential role in ZGA.

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.

The zinc finger protein CLAMP promotes long-range chromatin interactions that mediate dosage compensation of the Drosophila male X-chromosome.

BACKGROUND: Drosophila dosage compensation is an important model system for defining how active chromatin domains are formed. The male-specific lethal dosage compensation complex (MSLc) increases transcript levels of genes along the length of the single male X-chromosome to equalize with that expressed from the two female X-chromosomes. The strongest binding sites for MSLc cluster together in three-dimensional space largely independent of MSLc because clustering occurs in both sexes. CLAMP, a non-sex specific, ubiquitous zinc finger protein, binds synergistically with MSLc to enrich the occupancy of both factors on the male X-chromosome. RESULTS: Here, we demonstrate that CLAMP promotes the observed three-dimensional clustering of MSLc binding sites. Moreover, the X-enriched CLAMP protein more strongly promotes longer-range three-dimensional interactions on the X-chromosome than autosomes. Genome-wide, CLAMP promotes three-dimensional interactions between active chromatin regions together with other insulator proteins. CONCLUSION: Overall, we define how long-range interactions which are modulated by a locally enriched ubiquitous transcription factor promote hyper-activation of the X-chromosome to mediate dosage compensation.

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.

Dosage Compensation: How to Be Compensated Or Not?

Diverse dosage compensation mechanisms have evolved across species to equalize gene expression between sexes and between the sex chromosomes and autosomes. New results show that two opposite modes of dosage compensation can occur within one species, the monarch butterfly.

Targeting of the Dosage-Compensated Male X-Chromosome during Early Drosophila Development.

Dosage compensation, which corrects for the imbalance in X-linked gene expression between XX females and XY males, represents a model for how genes are targeted for coordinated regulation. However, the mechanism by which dosage compensation complexes identify the X chromosome during early development remains unknown because of the difficulty of sexing embryos before zygotic transcription using X- or Y-linked reporter transgenes. We used meiotic drive to sex Drosophila embryos before zygotic transcription and ChIP-seq to measure the dynamics of dosage compensation factor targeting. The Drosophila male-specific lethal dosage compensation complex (MSLc) requires the ubiquitous zinc-finger protein chromatin-linked adaptor for MSL proteins (CLAMP) to identify the X chromosome. We observe a multi-stage process in which MSLc first identifies CLAMP binding sites throughout the genome, followed by concentration at the strongest X-linked MSLc sites. We provide insight into the dynamics of binding site recognition by a large transcription complex during early development.

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.

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.

Differential Occupancy of Two GA-Binding Proteins Promotes Targeting of the Drosophila Dosage Compensation Complex to the Male X Chromosome.

Little is known about how variation in sequence composition alters transcription factor occupancy to precisely recruit large transcription complexes. A key model for understanding how transcription complexes are targeted is the Drosophila dosage compensation system in which the male-specific lethal (MSL) transcription complex specifically identifies and regulates the male X chromosome. The chromatin-linked adaptor for MSL proteins (CLAMP) zinc-finger protein targets MSL to the X chromosome but also binds to GA-rich sequence elements throughout the genome. Furthermore, the GAGA-associated factor (GAF) transcription factor also recognizes GA-rich sequences but does not associate with the MSL complex. Here, we demonstrate that MSL complex recruitment sites are optimal CLAMP targets. Specificity for CLAMP binding versus GAF binding is driven by variability in sequence composition within similar GA-rich motifs. Therefore, variation within seemingly similar cis elements drives the context-specific targeting of a large transcription complex.

The Drosophila CLAMP protein associates with diverse proteins on chromatin.

Gaining new insights into gene regulation involves an in-depth understanding of protein-protein interactions on chromatin. A powerful model for studying mechanisms of gene regulation is dosage compensation, a process that targets the X-chromosome to equalize gene expression between XY males and XX females. We previously identified a zinc finger protein in Drosophila melanogaster that plays a sex-specific role in targeting the Male-specific lethal (MSL) dosage compensation complex to the male X-chromosome, called the Chromatin-Linked Adapter for MSL Proteins (CLAMP). More recently, we established that CLAMP has non-sex-specific roles as an essential protein that regulates chromatin accessibility at promoters genome-wide. To identify associations between CLAMP and other factors in both male and female cells, we used two complementary mass spectrometry approaches. We demonstrate that CLAMP associates with the transcriptional regulator complex Negative Elongation Factor (NELF) in both sexes and determine that CLAMP reduces NELF recruitment to several target genes. In sum, we have identified many new CLAMP-associated factors and provide a resource for further study of this little understood essential protein.

Enhanced chromatin accessibility of the dosage compensated Drosophila male X-chromosome requires the CLAMP zinc finger protein.

The essential process of dosage compensation is required to equalize gene expression of X-chromosome genes between males (XY) and females (XX). In Drosophila, the conserved Male-specific lethal (MSL) histone acetyltransferase complex mediates dosage compensation by increasing transcript levels from genes on the single male X-chromosome approximately two-fold. Consistent with its increased levels of transcription, the male X-chromosome has enhanced chromatin accessibility, distinguishing it from the autosomes. Here, we demonstrate that the non-sex-specific CLAMP (Chromatin-linked adaptor for MSL proteins) zinc finger protein that recognizes GA-rich sequences genome-wide promotes the specialized chromatin environment on the male X-chromosome and can act over long genomic distances (~14 kb). Although MSL complex is required for increasing transcript levels of X-linked genes, it is not required for enhancing global male X-chromosome chromatin accessibility, and instead works cooperatively with CLAMP to facilitate an accessible chromatin configuration at its sites of highest occupancy. Furthermore, CLAMP regulates chromatin structure at strong MSL complex binding sites through promoting recruitment of the Nucleosome Remodeling Factor (NURF) complex. In contrast to the X-chromosome, CLAMP regulates chromatin and gene expression on autosomes through a distinct mechanism that does not involve NURF recruitment. Overall, our results support a model where synergy between a non-sex-specific transcription factor (CLAMP) and a sex-specific cofactor (MSL) creates a specialized chromatin domain on the male X-chromosome.

Drosophila Dosage Compensation Loci Associate with a Boundary-Forming Insulator Complex.

Chromatin entry sites (CES) are 100- to 1,500-bp elements that recruit male-specific lethal (MSL) complexes to the X chromosome to upregulate expression of X-linked genes in male flies. CES contain one or more ∼20-bp GA-rich sequences called MSL recognition elements (MREs) that are critical for dosage compensation. Recent studies indicate that CES also correspond to boundaries of X-chromosomal topologically associated domains (TADs). Here, we show that an ∼1,000-kDa complex called the late boundary complex (LBC), which is required for the functioning of the Bithorax complex boundary Fab-7, interacts specifically with a special class of CES that contain multiple MREs. Mutations in the MRE sequences of three of these CES that disrupt function in vivo abrogate interactions with the LBC. Moreover, reducing the levels of two LBC components compromises MSL recruitment. Finally, we show that several of the CES that are physically linked to each other in vivo are LBC interactors.

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.

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.