The histone chaperone NASP maintains H3-H4 reservoirs in the early Drosophila embryo.

Histones are essential for chromatin packaging, and histone supply must be tightly regulated as excess histones are toxic. To drive the rapid cell cycles of the early embryo, however, excess histones are maternally deposited. Therefore, soluble histones must be buffered by histone chaperones, but the chaperone necessary to stabilize soluble H3-H4 pools in the Drosophila embryo has yet to be identified. Here, we show that CG8223, the Drosophila homolog of NASP, is a H3-H4-specific chaperone in the early embryo. We demonstrate that, while a NASP null mutant is viable in Drosophila, NASP is a maternal effect gene. Embryos laid by NASP mutant mothers have a reduced rate of hatching and show defects in early embryogenesis. Critically, soluble H3-H4 pools are degraded in embryos laid by NASP mutant mothers. Our work identifies NASP as the critical H3-H4 histone chaperone in the Drosophila embryo.

BRWD3 promotes KDM5 degradation to maintain H3K4 methylation levels.

Histone modifications are critical for regulating chromatin structure and gene expression. Dysregulation of histone modifications likely contributes to disease states and cancer. Depletion of the chromatin-binding protein BRWD3 (Bromodomain and WD repeat-containing protein 3), a known substrate-specificity factor of the Cul4-DDB1 E3 ubiquitin ligase complex, results in increased H3K4me1 (H3 lysine 4 monomethylation) levels. The underlying mechanism linking BRWD3 and H3K4 methylation, however, has yet to be defined. Here, we show that depleting BRWD3 not only causes an increase in H3K4me1 levels but also causes a decrease in H3K4me3 (H3 lysine 4 trimethylation) levels, indicating that BRWD3 influences H3K4 methylation more broadly. Using immunoprecipitation coupled to quantitative mass spectrometry, we identified an interaction between BRWD3 and the H3K4-specific lysine demethylase 5 (KDM5/Lid), an enzyme that removes tri- and dimethyl marks from H3K4. Moreover, analysis of ChIP-seq (chromatin immunoprecipitation sequencing) data revealed that BRWD3 and KDM5 are significantly colocalized throughout the genome and H3K4me3 are highly enriched at BRWD3 binding sites. We show that BRWD3 promotes K48-linked polyubiquitination and degradation of KDM5 and that KDM5 degradation is dependent on both BRWD3 and Cul4. Critically, depleting KDM5 fully restores altered H3K4me3 levels and partially restores H3K4me1 levels upon BRWD3 depletion. Together, our results demonstrate that BRWD3 regulates KDM5 activity to balance H3K4 methylation levels.

Integrative analysis of gene amplification in Drosophila follicle cells: parameters of origin activation and repression.

In metazoans, how replication origins are specified and subsequently activated is not well understood. Drosophila amplicons in follicle cells (DAFCs) are genomic regions that undergo rereplication to increase DNA copy number. We identified all DAFCs by comparative genomic hybridization, uncovering two new amplicons in addition to four known previously. The complete identification of all DAFCs enabled us to investigate these in vivo replicons with respect to parameters of transcription, localization of the origin recognition complex (ORC), and histone acetylation, yielding important insights into gene amplification as a metazoan replication model. Significantly, ORC is bound across domains spanning 10 or more kilobases at the DAFC rather than at a specific site. Additionally, ORC is bound at many regions that do not undergo amplification, and, in contrast to cell culture, these regions do not correlate with high gene expression. As a developmental strategy, gene amplification is not the predominant means of achieving high expression levels, even in cells capable of amplification. Intriguingly, we found that, in some strains, a new amplicon, DAFC-22B, does not amplify, a consequence of distant repression of ORC binding and origin activation. This repression is alleviated when a fragment containing the origin is placed in different genomic contexts.

Understanding replication fork progression, stability, and chromosome fragility by exploiting the Suppressor of Underreplication protein.

There are many layers of regulation governing DNA replication to ensure that genetic information is accurately transmitted from mother cell to daughter cell. While much of the control occurs at the level of origin selection and firing, less is known about how replication fork progression is controlled throughout the genome. In Drosophila polytene cells, specific regions of the genome become repressed for DNA replication, resulting in underreplication and decreased copy number. Importantly, underreplicated domains share properties with common fragile sites. The Suppressor of Underreplication protein SUUR is essential for this repression. Recent work established that SUUR functions by directly inhibiting replication fork progression, raising several interesting questions as to how replication fork progression and stability can be modulated within targeted regions of the genome. Here we discuss potential mechanisms by which replication fork inhibition can be achieved and the consequences this has on genome stability and copy number control.

Developmental control of gene copy number by repression of replication initiation and fork progression.

Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands, and found they are transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of the origin recognition complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. Inhibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce copy number. Although repressive histone marks are removed when SUUR is mutated and copy number restored, neither transcription nor ORC binding is reinstated. Tethering of the SUUR protein to a specific site is insufficient to block replication, however. These results establish that developmental control of DNA replication, at both the initiation and elongation stages, is a mechanism to change gene copy number during differentiation.

Regulation of DNA replication during development.

As development unfolds, DNA replication is not only coordinated with cell proliferation, but is regulated uniquely in specific cell types and organs. This differential regulation of DNA synthesis requires crosstalk between DNA replication and differentiation. This dynamic aspect of DNA replication is highlighted by the finding that the distribution of replication origins varies between differentiated cell types and changes with differentiation. Moreover, differential DNA replication in some cell types can lead to increases or decreases in gene copy number along chromosomes. This review highlights the recent advances and technologies that have provided us with new insights into the developmental regulation of DNA replication.

Developmental control of the DNA replication and transcription programs.

Polyploid or polytene cells, which have more than 2C DNA content, are widespread throughout nature and present in most differentiated Drosophila tissues. These cells also can display differential replication, that is, genomic regions of increased or decreased DNA copy number relative to overall genomic ploidy. How frequently differential replication is used as a developmental strategy remains unclear. Here, we use genome-wide array-based comparative genomic hybridization (aCGH) to profile differential DNA replication in isolated and purified larval fat body and midgut tissues of Drosophila, and we compare them with recent aCGH profiles of the larval salivary gland. We identify sites of euchromatic underreplication that are common to all three tissues and others that are tissue specific. We demonstrate that both common and tissue-specific underreplicated sites are dependent on the Suppressor of Underreplication protein, SUUR. mRNA-seq profiling shows that whereas underreplicated regions are generally transcriptionally silent in the larval midgut and salivary gland, transcriptional silencing and underreplication have been uncoupled in the larval fat body. In addition to revealing the prevalence of differential replication, our results show that transcriptional silencing and underreplication can be mechanistically uncoupled.

PCR cloning Intermediated Gibson assembly (PIG) for Constructing DNA Repair Templates in CRISPR-Cas9 Based Gene Editing.

The CRISPR-Cas9 gene-editing system has revolutionized genome engineering, allowing precise modifications to be made in a wide range of organisms. One significant challenge associated with CRISPR-Cas9 mediated gene editing is the construction of DNA repair templates containing homology arms, a screenable marker and a tag sequence of interest. Here, we present an efficient, two-step strategy to generate DNA repair templates in approximately one week, facilitating rapid and precise genome engineering applications.

BRWD3 promotes KDM5 degradation to maintain H3K4 methylation levels.

Histone modifications are critical for regulating chromatin structure and gene expression. Dysregulation of histone modifications likely contributes to disease states and cancer. Depletion of the chromatin-binding protein BRWD3, a known substrate-specificity factor of the Cul4-DDB1 E3 ubiquitin ligase complex, results in increased in H3K4me1 levels. The underlying mechanism linking BRWD3 and H3K4 methylation, however, has yet to be defined. Here, we show that depleting BRWD3 not only causes an increase in H3K4me1 levels, but also causes a decrease in H3K4me3 levels, indicating that BRWD3 influences H3K4 methylation more broadly. Using immunoprecipitation coupled to quantitative mass spectrometry, we identified an interaction between BRWD3 and the H3K4-specific demethylase 5 (KDM5/Lid), an enzyme that removes tri- and di- methyl marks from H3K4. Moreover, analysis of ChIP-seq data revealed that BRWD3 and KDM5 are significantly co- localized throughout the genome and that sites of H3K4me3 are highly enriched at BRWD3 binding sites. We show that BRWD3 promotes K48-linked polyubiquitination and degradation of KDM5 and that KDM5 degradation is dependent on both BRWD3 and Cul4. Critically, depleting KDM5 fully restores altered H3K4me3 levels and partially restores H3K4me1 levels upon BRWD3 depletion. Together, our results demonstrate that BRWD3 regulates KDM5 activity to balance H3K4 methylation levels.

Rif1-Dependent Control of Replication Timing.

Successful duplication of the genome requires the accurate replication of billions of base pairs of DNA within a relatively short time frame. Failure to accurately replicate the genome results in genomic instability and a host of diseases. To faithfully and rapidly replicate the genome, DNA replication must be tightly regulated and coordinated with many other nuclear processes. These regulations, however, must also be flexible as replication kinetics can change through development and differentiation. Exactly how DNA replication is regulated and how this regulation changes through development is an active field of research. One aspect of genome duplication where much remains to be discovered is replication timing (RT), which dictates when each segment of the genome is replicated during S phase. All organisms display some level of RT, yet the precise mechanisms that govern RT remain are not fully understood. The study of Rif1, a protein that actively regulates RT from yeast to humans, provides a key to unlock the underlying molecular mechanisms controlling RT. The paradigm for Rif1 function is to delay helicase activation within certain regions of the genome, causing these regions to replicate late in S phase. Many questions, however, remain about the intricacies of Rif1 function. Here, we review the current models for the activity of Rif1 with the goal of trying to understand how Rif1 functions to establish the RT program.

R-loop Mapping and Characterization During Drosophila Embryogenesis Reveals Developmental Plasticity in R-loop Signatures.

R-loops are involved in transcriptional regulation, DNA and histone post-translational modifications, genome replication and genome stability. To what extent R-loop abundance and genome-wide localization is actively regulated during metazoan embryogenesis is unknown. Drosophila embryogenesis provides a powerful system to address these questions due to its well-characterized developmental program, the sudden onset of zygotic transcription and available genome-wide data sets. Here, we measure the overall abundance and genome localization of R-loops in early and late-stage embryos relative to Drosophila cultured cells. We demonstrate that absolute R-loop levels change during embryogenesis and that RNaseH1 catalytic activity is critical for embryonic development. R-loop mapping by strand-specific DRIP-seq reveals that R-loop localization is plastic across development, both in the genes which form R-loops and where they localize relative to gene bodies. Importantly, these changes are not driven by changes in the transcriptional program. Negative GC skew and absolute changes in AT skew are associated with R-loop formation in Drosophila. Furthermore, we demonstrate that while some chromatin binding proteins and histone modifications such as H3K27me3 are associated with R-loops throughout development, other chromatin factors associated with R-loops in a developmental specific manner. Our findings highlight the importance and developmental plasticity of R-loops during Drosophila embryogenesis.

Identification of replication fork-associated proteins in Drosophila embryos and cultured cells using iPOND coupled to quantitative mass spectrometry.

Replication of the eukaryotic genome requires the formation of thousands of replication forks that must work in concert to accurately replicate the genetic and epigenetic information. Defining replication fork-associated proteins is a key step in understanding how genomes are replicated and repaired in the context of chromatin to maintain genome stability. To identify replication fork-associated proteins, we performed iPOND (Isolation of Proteins on Nascent DNA) coupled to quantitative mass spectrometry in Drosophila embryos and cultured cells. We identified 76 and 278 fork-associated proteins in post-MZT embryos and Drosophila cultured S2 cells, respectively. By performing a targeted screen of a subset of these proteins, we demonstrate that BRWD3, a targeting specificity factor for the DDB1/Cul4 ubiquitin ligase complex (CRL4), functions at or in close proximity to replication forks to promote fork progression and maintain genome stability. Altogether, our work provides a valuable resource for those interested in DNA replication, repair and chromatin assembly during development.

Nucleoporins facilitate ORC loading onto chromatin.

The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we perform immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we find that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis reveals that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, decreases ORC2 loading onto chromatin. Depleting Elys also sensitizes cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a connection between ORC, replication initiation, and nucleoporins, suggesting a function for nucleoporins in metazoan replication initiation.

Escherichia coli nucleoside diphosphate kinase mutants depend on translesion DNA synthesis to prevent mutagenesis.

Escherichia coli nucleoside diphosphate (NDP) kinase mutants have an increased frequency of spontaneous mutation, possibly due to uracil misincorporation into DNA. Here we show that NDP kinase mutants are dependent on translesion DNA synthesis, often a mutagenic form of DNA synthesis, to prevent mutagenesis.

The relationship between dNTP pool levels and mutagenesis in an Escherichia coli NDP kinase mutant.

Loss of nucleoside diphosphate kinase (Ndk) function in Escherichia coli results in an increased frequency of spontaneous mutation and an imbalance in dNTP pool levels. It is presumed that the imbalance in dNTP pool levels is responsible for the mutator phenotype of an E. coli ndk mutant. A human homologue of Ndk and potential suppressor of tumor metastasis, nm23-H2, can complement the mutagenic phenotype of an E. coli ndk mutant. Here, we show that the antimutagenic property of nm23-H2 in E. coli is independent of dNTP pool levels, indicating that dNTP pool imbalance is not responsible for the mutator phenotype associated with the loss of ndk function. We have identified multiple genetic interactions between ndk and genes involved in the metabolism of dUTP, a potentially mutagenic precursor of thymidine biosynthesis. We show that loss of ndk function is synergistic with a dut-1 mutation and synthetically lethal with the loss of thymidine kinase function. Our results suggest that Ndk prevents the accumulation of dUTP in vivo. Based on these results and biochemical studies of Ndk, we propose that the mutagenic phenotype of an ndk mutant is caused by excess misincorporation of uracil in place of thymidine combined with a defect in the uracil base excision pathway.

Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila.

Regulation of DNA replication and copy number is necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rap1-interacting factor 1 (Rif1) is a key regulator of RT and has a critical function in copy number control in polyploid cells. Previously, we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 acts to promote late replication, which is necessary for SUUR-dependent underreplication. Our work provides new insight into the process of UR and its links to RT.

Rif1 Functions in a Tissue-Specific Manner To Control Replication Timing Through Its PP1-Binding Motif.

Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early- and late-replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, we utilized the powerful developmental systems available in Drosophila melanogaster We generated and compared RT profiles from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. Our results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, we demonstrate that the RT differences we observed in all cases are largely independent of transcriptional differences. We also employed a genetic approach in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. Our results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, our data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs.

Rif1 inhibits replication fork progression and controls DNA copy number in Drosophila.

Control of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila polyploid cells, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR's SNF2 domain, we identified an interaction between SUUR and Rif1. Rif1 has many roles in DNA metabolism and regulates the replication timing program. We demonstrate that repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in a partially SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. Our findings uncover an unrecognized function of the Rif1 protein as a regulator of replication fork progression.

DNA copy-number control through inhibition of replication fork progression.

Proper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell-cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein SUUR through an unknown mechanism. We demonstrate that SUUR is recruited to active replication forks and mediates the repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass spectrometry identification of SUUR-associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. Our results reveal that control of eukaryotic DNA copy number can occur through the inhibition of replication fork progression.

Identification of functional elements and regulatory circuits by Drosophila modENCODE.

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.