Sequence reliance of the Drosophila context-dependent transcription factor CLAMP.

Despite binding similar cis elements in multiple locations, a single transcription factor (TF) often performs context-dependent functions at different loci. How factors integrate cis sequence and genomic context is still poorly understood and has implications for off-target effects in genetic engineering. The Drosophila context-dependent TF chromatin-linked adaptor for male-specific lethal proteins (CLAMP) targets similar GA-rich cis elements on the X-chromosome and at the histone gene locus but recruits very different, locus-specific factors. We discover that CLAMP leverages information from both cis element and local sequence to perform context-specific functions. Our observations imply the importance of other cues, including protein-protein interactions and the presence of additional cofactors.

Sequence reliance of a Drosophila context-dependent transcription factor.

Despite binding similar cis elements in multiple locations, a single transcription factor often performs context-dependent functions at different loci. How factors integrate cis sequence and genomic context is still poorly understood and has implications for off-target effects in genetic engineering. The Drosophila context-dependent transcription factor CLAMP targets similar GA-rich cis elements on the X-chromosome and at the histone gene locus but recruits very different, loci-specific factors. We discover that CLAMP leverages information from both cis element and local sequence to perform context-specific functions. Our observations imply the importance of other cues, including protein-protein interactions and the presence of additional cofactors.

A cost-free CURE: using bioinformatics to identify DNA-binding factors at a specific genomic locus.

Research experiences provide diverse benefits for undergraduates. Many academic institutions have adopted course-based undergraduate research experiences (CUREs) to improve student access to research opportunities. However, potential instructors of a CURE might still face financial or practical hurdles that prevent implementation. Bioinformatics research offers an alternative that is free, safe, compatible with remote learning, and may be more accessible for students with disabilities. Here, we describe a bioinformatics CURE that leverages publicly available datasets to discover novel proteins that target an instructor-determined genomic locus of interest. We use the free, user-friendly bioinformatics platform Galaxy to map ChIP-seq datasets to a genome, which removes the computing burden from students. Both faculty and students directly benefit from this CURE, as faculty can perform candidate screens and publish CURE results. Students gain not only basic bioinformatics knowledge, but also transferable skills, including scientific communication, database navigation, and primary literature experience. The CURE is flexible and can be expanded to analyze different types of high-throughput data or to investigate different genomic loci in any species.

A hybrid RNA FISH immunofluorescence protocol on Drosophila polytene chromosomes.

OBJECTIVES: Investigating protein-DNA interactions is imperative to understanding fundamental concepts such as cell growth, differentiation, and cell development in many systems. Sequencing techniques such as ChIP-seq can yield genome-wide DNA binding profiles of transcription factors; however this assay can be expensive, time-consuming, may not be informative for repetitive regions of the genome, and depend heavily upon antibody suitability. Combining DNA fluorescence in situ hybridization (FISH) with immunofluorescence (IF) is a quicker and inexpensive approach which has historically been used to investigate protein-DNA interactions in individual nuclei. However, these assays are sometimes incompatible due to the required denaturation step in DNA FISH that can alter protein epitopes, hindering primary antibody binding. Additionally, combining DNA FISH with IF may be challenging for less experienced trainees. Our goal was to develop an alternative technique to investigate protein-DNA interactions by combining RNA FISH with IF. RESULTS: We developed a hybrid RNA FISH-IF protocol for use on Drosophila melanogaster polytene chromosome spreads in order to visualize colocalization of proteins and DNA loci. We demonstrate that this assay is sensitive enough to determine if our protein of interest, Multi sex combs (Mxc), localizes to single-copy target transgenes carrying histone genes. Overall, this study provides an alternative, accessible method for investigating protein-DNA interactions at the single gene level in Drosophila melanogaster polytene chromosomes.

A bioinformatics screen reveals hox and chromatin remodeling factors at the Drosophila histone locus.

BACKGROUND: Cells orchestrate histone biogenesis with strict temporal and quantitative control. To efficiently regulate histone biogenesis, the repetitive Drosophila melanogaster replication-dependent histone genes are arrayed and clustered at a single locus. Regulatory factors concentrate in a nuclear body known as the histone locus body (HLB), which forms around the locus. Historically, HLB factors are largely discovered by chance, and few are known to interact directly with DNA. It is therefore unclear how the histone genes are specifically targeted for unique and coordinated regulation. RESULTS: To expand the list of known HLB factors, we performed a candidate-based screen by mapping 30 publicly available ChIP datasets of 27 unique factors to the Drosophila histone gene array. We identified novel transcription factor candidates, including the Drosophila Hox proteins Ultrabithorax (Ubx), Abdominal-A (Abd-A), and Abdominal-B (Abd-B), suggesting a new pathway for these factors in influencing body plan morphogenesis. Additionally, we identified six other factors that target the histone gene array: JIL-1, hormone-like receptor 78 (Hr78), the long isoform of female sterile homeotic (1) (fs(1)h) as well as the general transcription factors TBP associated factor 1 (TAF-1), Transcription Factor IIB (TFIIB), and Transcription Factor IIF (TFIIF). CONCLUSIONS: Our foundational screen provides several candidates for future studies into factors that may influence histone biogenesis. Further, our study emphasizes the powerful reservoir of publicly available datasets, which can be mined as a primary screening technique.

A hybrid RNA FISH immunofluorescence protocol on Drosophila polytene chromosomes.

Objectives: Investigating protein-DNA interactions is imperative to understanding fundamental concepts such as cell growth, differentiation, and cell development in many systems. Sequencing techniques such as ChIP-seq can yield genome-wide DNA binding profiles of transcription factors; however this assay can be expensive, time-consuming, may not be informative for repetitive regions of the genome, and depend heavily upon antibody suitability. Combining DNA fluorescence in situ hybridization (FISH) with immunofluorescence (IF) is a quicker and inexpensive approach which has historically been used to investigate protein-DNA interactions in individual nuclei. However, these assays are sometimes incompatible due to the required denaturation step in DNA FISH that can alter protein epitopes, hindering primary antibody binding. Additionally, combining DNA FISH with IF may be challenging for less experienced trainees. Our goal was to develop an alternative technique to investigate protein-DNA interactions by combining RNA FISH with IF. Results: We developed a hybrid RNA FISH and IF protocol for use on Drosophila melanogaster polytene chromosome spreads in order to visualize colocalization of proteins and DNA loci. We demonstrate that this assay is sensitive enough to determine if our protein of interest, Multi-sex combs (Mxc), localizes to single-copy target transgenes carrying histone genes. Overall, this study provides an alternative, accessible method for investigating protein-DNA interactions at the single gene level in Drosophila melanogaster polytene chromosomes.

A bioinformatics screen reveals Hox and chromatin remodeling factors at the Drosophila histone locus.

Cells orchestrate histone biogenesis with strict temporal and quantitative control. To efficiently regulate histone biogenesis, the repetitive Drosophila melanogaster replication-dependent histone genes are arrayed and clustered at a single locus. Regulatory factors concentrate in a nuclear body known as the histone locus body (HLB), which forms around the locus. Historically, HLB factors are largely discovered by chance, and few are known to interact directly with DNA. It is therefore unclear how the histone genes are specifically targeted for unique and coordinated regulation. To expand the list of known HLB factors, we performed a candidate-based screen by mapping 30 publicly available ChIP datasets and 27 factors to the Drosophila histone gene array. We identified novel transcription factor candidates, including the Drosophila Hox proteins Ultrabithorax, Abdominal-A and Abdominal-B, suggesting a new pathway for these factors in influencing body plan morphogenesis. Additionally, we identified six other transcription factors that target the histone gene array: JIL-1, Hr78, the long isoform of fs(1)h as well as the generalized transcription factors TAF-1, TFIIB, and TFIIF. Our foundational screen provides several candidates for future studies into factors that may influence histone biogenesis. Further, our study emphasizes the powerful reservoir of publicly available datasets, which can be mined as a primary screening technique.

Zelda is dispensable for Drosophila melanogaster histone gene regulation.

To ensure that the embryo can package exponentially increasing amounts of DNA, replication-dependent histones are some of the earliest transcribed genes from the zygotic genome. However, how the histone genes are identified is not known. The pioneer factors Zelda and CLAMP collaborate at a subset of genes to regulate zygotic genome activation in Drosophila melanogaster and target early activated genes to induce transcription. CLAMP also regulates the embryonic histone genes and helps establish the histone locus body, a suite of factors that controls histone mRNA biosynthesis. The relationship between Zelda and CLAMP led us to hypothesize that Zelda helps identify histone genes for early embryonic expression. We found that Zelda targets the histone locus early during embryogenesis, prior to histone gene expression. However, depletion of zelda in the early embryo does not affect histone mRNA levels or histone locus body formation. While surprising, these results concur with other investigations into Zelda's role in the early embryo, suggesting the earliest factors responsible for specifying the zygotic histone genes remain undiscovered.

CHD1 controls H3.3 incorporation in adult brain chromatin to maintain metabolic homeostasis and normal lifespan.

The ATP-dependent chromatin remodeling factor CHD1 is essential for the assembly of variant histone H3.3 into paternal chromatin during sperm chromatin remodeling in fertilized eggs. It remains unclear, however, if CHD1 has a similar role in normal diploid cells. Using a specifically tailored quantitative mass spectrometry approach, we show that Chd1 disruption results in reduced H3.3 levels in heads of Chd1 mutant flies. Chd1 deletion perturbs brain chromatin structure in a similar way as H3.3 deletion and leads to global de-repression of transcription. The physiological consequences are reduced food intake, metabolic alterations, and shortened lifespan. Notably, brain-specific CHD1 expression rescues these phenotypes. We further demonstrate a strong genetic interaction between Chd1 and H3.3 chaperone Hira. Thus, our findings establish CHD1 as a factor required for the assembly of H3.3-containing chromatin in adult cells and suggest a crucial role for CHD1 in the brain as a regulator of organismal health and longevity.

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.

Drosophila histone locus body assembly and function involves multiple interactions.

The histone locus body (HLB) assembles at replication-dependent (RD) histone loci and concentrates factors required for RD histone mRNA biosynthesis. The Drosophila melanogaster genome has a single locus comprised of ∼100 copies of a tandemly arrayed 5-kB repeat unit containing one copy of each of the 5 RD histone genes. To determine sequence elements required for D. melanogaster HLB formation and histone gene expression, we used transgenic gene arrays containing 12 copies of the histone repeat unit that functionally complement loss of the ∼200 endogenous RD histone genes. A 12x histone gene array in which all H3-H4 promoters were replaced with H2a-H2b promoters (12xPR) does not form an HLB or express high levels of RD histone mRNA in the presence of the endogenous histone genes. In contrast, this same transgenic array is active in HLB assembly and RD histone gene expression in the absence of the endogenous RD histone genes and rescues the lethality caused by homozygous deletion of the RD histone locus. The HLB formed in the absence of endogenous RD histone genes on the mutant 12x array contains all known factors present in the wild-type HLB including CLAMP, which normally binds to GAGA repeats in the H3-H4 promoter. These data suggest that multiple protein-protein and/or protein-DNA interactions contribute to HLB formation, and that the large number of endogenous RD histone gene copies sequester available factor(s) from attenuated transgenic arrays, thereby preventing HLB formation and gene expression on these arrays.

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.

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.

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.

Dynamic response of RNA editing to temperature in Drosophila.

BACKGROUND: Adenosine-to-inosine RNA editing is a highly conserved process that post-transcriptionally modifies mRNA, generating proteomic diversity, particularly within the nervous system of metazoans. Transcripts encoding proteins involved in neurotransmission predominate as targets of such modifications. Previous reports suggest that RNA editing is responsive to environmental inputs in the form of temperature alterations. However, the molecular determinants underlying temperature-dependent RNA editing responses are not well understood. RESULTS: Using the poikilotherm Drosophila, we show that acute temperature alterations within a normal physiological range result in substantial changes in RNA editing levels. Our examination of particular sites reveals diversity in the patterns with which editing responds to temperature, and these patterns are conserved across five species of Drosophilidae representing over 10 million years of divergence. In addition, we show that expression of the editing enzyme, ADAR (adenosine deaminase acting on RNA), is dramatically decreased at elevated temperatures, partially, but not fully, explaining some target responses to temperature. Interestingly, this reduction in editing enzyme levels at elevated temperature is only partially reversed by a return to lower temperatures. Lastly, we show that engineered structural variants of the most temperature-sensitive editing site, in a sodium channel transcript, perturb thermal responsiveness in RNA editing profile for a particular RNA structure. CONCLUSIONS: Our results suggest that the RNA editing process responds to temperature alterations via two distinct molecular mechanisms: through intrinsic thermo-sensitivity of the RNA structures that direct editing, and due to temperature sensitive expression or stability of the RNA editing enzyme. Environmental cues, in this case temperature, rapidly reprogram the Drosophila transcriptome through RNA editing, presumably resulting in altered proteomic ratios of edited and unedited proteins.

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.

Tertiary structural elements determine the extent and specificity of messenger RNA editing.

The specificity and extent of RNA editing by ADAR enzymes is determined largely by local primary sequence and secondary structural imperfections in duplex RNA. Here we surgically alter conserved cis elements associated with a cluster of ADAR modification sites within the endogenous Drosophila paralytic transcript. In addition to the local requirement for a central imperfect RNA duplex containing the modified adenosines, we demonstrate that a secondary RNA duplex containing splicing signals strongly modulates RNA editing. A subtle non-coding mutation, extending base pairing of this accessory helix, confers significant phenotypic consequences via effects on splicing. Through mutation/counter-mutation, we also uncover and functionally replace a highly conserved intronic long-range tertiary pseudoknot that is absolutely required for deamination of one particular adenosine in the central duplex. Our results demonstrate that complex RNA tertiary structures, which may be difficult to predict computationally, form in vivo and can regulate RNA-editing events.

The ADAR protein family.

Adenosine to inosine (A-to-I) RNA editing is a post-transcriptional process by which adenosines are selectively converted to inosines in double-stranded RNA (dsRNA) substrates. A highly conserved group of enzymes, the adenosine deaminase acting on RNA (ADAR) family, mediates this reaction. All ADARs share a common domain architecture consisting of a variable number of amino-terminal dsRNA binding domains (dsRBDs) and a carboxy-terminal catalytic deaminase domain. ADAR family members are highly expressed in the metazoan nervous system, where these enzymes predominantly localize to the neuronal nucleus. Once in the nucleus, ADARs participate in the modification of specific adenosines in pre-mRNAs of proteins involved in electrical and chemical neurotransmission, including pre-synaptic release machineries, and voltage- and ligand-gated ion channels. Most RNA editing sites in these nervous system targets result in non-synonymous codon changes in functionally important, usually conserved, residues and RNA editing deficiencies in various model organisms bear out a crucial role for ADARs in nervous system function. Mutation or deletion of ADAR genes results in striking phenotypes, including seizure episodes, extreme uncoordination, and neurodegeneration. Not only does the process of RNA editing alter important nervous system peptides, but ADARs also regulate gene expression through modification of dsRNA substrates that enter the RNA interference (RNAi) pathway and may then act at the chromatin level. Here, we present a review on the current knowledge regarding the ADAR protein family, including evolutionary history, key structural features, localization, function and mechanism.

The intricate relationship between RNA structure, editing, and splicing.

Post-transcriptional modifications such as RNA editing and splicing diversify the proteome while limiting the necessary size of the genome. Although splicing globally rearranges existing information within the transcript, the conserved process of adenosine-to-inosine RNA editing recodes the message through single nucleotide changes, often at very specific locations. Because inosine is interpreted as guanosine by the cellular machineries, editing effectively results in the substitution of a guanosine for an adenosine in the primary RNA sequence. Precise control of editing is dictated by duplex structures in the transcript, formed between the exonic region surrounding the editing site and cis regulatory elements often localized in a nearby intron, suggesting that editing must precede splicing. However, the precise relationship between these post-transcriptional processes remains unclear. Here we present general commonalities of RNA editing substrates and consequential predictions regarding the interaction between editing and splicing. We also discuss anomalies and interesting cases of RNA editing that confound our understanding of the relationship between these post-transcriptional processes.