Tissue-Specific DNA Replication Defects in Drosophila melanogaster Caused by a Meier-Gorlin Syndrome Mutation in Orc4.

Meier-Gorlin syndrome is a rare recessive disorder characterized by a number of distinct tissue-specific developmental defects. Genes encoding members of the origin recognition complex (ORC) and additional proteins essential for DNA replication (CDC6, CDT1, GMNN, CDC45, MCM5, and DONSON) are mutated in individuals diagnosed with MGS. The essential role of ORC is to license origins during the G1 phase of the cell cycle, but ORC has also been implicated in several nonreplicative functions. Because of its essential role in DNA replication, ORC is required for every cell division during development. Thus, it is unclear how the Meier-Gorlin syndrome mutations in genes encoding ORC lead to the tissue-specific defects associated with the disease. To begin to address these issues, we used Cas9-mediated genome engineering to generate a Drosophila melanogaster model of individuals carrying a specific Meier-Gorlin syndrome mutation in ORC4 along with control strains. Together these strains provide the first metazoan model for an MGS mutation in which the mutation was engineered at the endogenous locus along with precisely defined control strains. Flies homozygous for the engineered MGS allele reach adulthood, but with several tissue-specific defects. Genetic analysis revealed that this Orc4 allele was a hypomorph. Mutant females were sterile, and phenotypic analyses suggested that defects in DNA replication was an underlying cause. By leveraging the well-studied Drosophila system, we provide evidence that a disease-causing mutation in Orc4 disrupts DNA replication, and we propose that in individuals with MGS defects arise preferentially in tissues with a high-replication demand.

Continued Activity of the Pioneer Factor Zelda Is Required to Drive Zygotic Genome Activation.

Reprogramming cell fate during the first stages of embryogenesis requires that transcriptional activators gain access to the genome and remodel the zygotic transcriptome. Nonetheless, it is not clear whether the continued activity of these pioneering factors is required throughout zygotic genome activation or whether they are only required early to establish cis-regulatory regions. To address this question, we developed an optogenetic strategy to rapidly and reversibly inactivate the master regulator of genome activation in Drosophila, Zelda. Using this strategy, we demonstrate that continued Zelda activity is required throughout genome activation. We show that Zelda binds DNA in the context of nucleosomes and suggest that this allows Zelda to occupy the genome despite the rapid division cycles in the early embryo. These data identify a powerful strategy to inactivate transcription factor function during development and suggest that reprogramming in the embryo may require specific, continuous pioneering functions to activate the genome.

Optogenetic Inactivation of Transcription Factors in the Early Embryo of Drosophila.

The early embryo of Drosophila melanogaster exists as a rapidly dividing syncytium of nuclei that are transcriptionally silent. Maternally deposited factors are required to awaken the genome and assist in the transition from maternal to zygotic control of development. Because many of these essential factors are maternally deposited and the early nuclear divisions are so rapid, it has been difficult to assess the functional role of transcription factors at discrete points in early embryonic development. To address this issue, we have developed an optogenetic system that can rapidly and reversibly inactivate transcription factors with nuclear-cycle resolution. The temporal precision enabled by this technique will allow a mechanistic understanding of how transcription factors function together to control genome activation and patterning in the early embryo and is likely broadly applicable to factors throughout embryogenesis.

H3K36 Methylation Regulates Nutrient Stress Response in Saccharomyces cerevisiae by Enforcing Transcriptional Fidelity.

Set2-mediated histone methylation at H3K36 regulates diverse activities, including DNA repair, mRNA splicing, and suppression of inappropriate (cryptic) transcription. Although failure of Set2 to suppress cryptic transcription has been linked to decreased lifespan, the extent to which cryptic transcription influences other cellular functions is poorly understood. Here, we uncover a role for H3K36 methylation in the regulation of the nutrient stress response pathway. We found that the transcriptional response to nutrient stress was dysregulated in SET2-deleted (set2Δ) cells and was correlated with genome-wide bi-directional cryptic transcription that originated from within gene bodies. Antisense transcripts arising from these cryptic events extended into the promoters of the genes from which they arose and were associated with decreased sense transcription under nutrient stress conditions. These results suggest that Set2-enforced transcriptional fidelity is critical to the proper regulation of inducible and highly regulated transcription programs.

Shaping the cellular landscape with Set2/SETD2 methylation.

Chromatin structure is a major barrier to gene transcription that must be disrupted and re-set during each round of transcription. Central to this process is the Set2/SETD2 methyltransferase that mediates co-transcriptional methylation to histone H3 at lysine 36 (H3K36me). Studies reveal that H3K36me not only prevents inappropriate transcriptional initiation from arising within gene bodies, but that it has other conserved functions that include the repair of damaged DNA and regulation of pre-mRNA splicing. Consistent with the importance of Set2/SETD2 in chromatin biology, mutations of SETD2, or mutations at or near H3K36 in H3.3, have recently been found to underlie cancer development. This review will summarize the latest insights into the functions of Set2/SETD2 in genome regulation and cancer development.

DRH1, a p68-related RNA helicase gene, is required for chromosome breakage in Tetrahymena.

The p68 DEAD box helicases comprise a widely conserved protein family involved in a large range of biological processes including transcription, splicing and translation. The genome of the ciliate Tetrahymena thermophile encodes two p68-like helicases, Drh1p and Lia2p. We show that DRH1 is essential for growth and completion of development. In growing cells, Drh1p is excluded from the nucleus and accumulates near cortical basal bodies. In contrast, during sexual reproduction, this protein localizes to meiotic micronuclei, initially in punctate foci in regions where centromeres and telomeres are known to reside and later in post-zygotic differentiating somatic macronuclei. Differentiation of the macronuclear genome involves extensive DNA rearrangements including fragmentation of the five pairs of germline-derived chromosomes into 180 chromosomal sub-fragments that are stabilized by de novo telomere deletion. In addition, thousands of internal eliminated sequences (IESs) are excised from loci dispersed throughout the genome. Strains with DRH1 deleted from the germline nuclei, which do not express the protein during post-zygotic development, fail to fragment the developing macronuclear chromosomes. IES excision still occurs in the absence of DRH1 zygotic expression; thus, Drh1p is the first protein found to be specifically required for chromosome breakage but not DNA elimination.

Combinatorial Histone Readout by the Dual Plant Homeodomain (PHD) Fingers of Rco1 Mediates Rpd3S Chromatin Recruitment and the Maintenance of Transcriptional Fidelity.

The plant homeodomain (PHD) finger is found in many chromatin-associated proteins and functions to recruit effector proteins to chromatin through its ability to bind both methylated and unmethylated histone residues. Here, we show that the dual PHD fingers of Rco1, a member of the Rpd3S histone deacetylase complex recruited to transcribing genes, operate in a combinatorial manner in targeting the Rpd3S complex to histone H3 in chromatin. Although mutations in either the first or second PHD finger allow for Rpd3S complex formation, the assembled complexes from these mutants cannot recognize nucleosomes or function to maintain chromatin structure and prevent cryptic transcriptional initiation from within transcribed regions. Taken together, our findings establish a critical role of combinatorial readout in maintaining chromatin organization and in enforcing the transcriptional fidelity of genes.

Targeting of P-Element Reporters to Heterochromatic Domains by Transposable Element 1360 in Drosophila melanogaster.

Heterochromatin is a common DNA packaging form employed by eukaryotes to constitutively silence transposable elements. Determining which sequences to package as heterochromatin is vital for an organism. Here, we use Drosophila melanogaster to study heterochromatin formation, exploiting position-effect variegation, a process whereby a transgene is silenced stochastically if inserted in proximity to heterochromatin, leading to a variegating phenotype. Previous studies identified the transposable element 1360 as a target for heterochromatin formation. We use transgene reporters with either one or four copies of 1360 to determine if increasing local repeat density can alter the fraction of the genome supporting heterochromatin formation. We find that including 1360 in the reporter increases the frequency with which variegating phenotypes are observed. This increase is due to a greater recovery of insertions at the telomere-associated sequences (∼50% of variegating inserts). In contrast to variegating insertions elsewhere, the phenotype of telomere-associated sequence insertions is largely independent of the presence of 1360 in the reporter. We find that variegating and fully expressed transgenes are located in different types of chromatin and that variegating reporters in the telomere-associated sequences differ from those in pericentric heterochromatin. Indeed, chromatin marks at the transgene insertion site can be used to predict the eye phenotype. Our analysis reveals that increasing the local repeat density (via the transgene reporter) does not enlarge the fraction of the genome supporting heterochromatin formation. Rather, additional copies of 1360 appear to target the reporter to the telomere-associated sequences with greater efficiency, thus leading to an increased recovery of variegating insertions.

Reprint of: Nitric oxide-releasing polysaccharide derivative exhibits 8-log reduction against Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus.

Health-care associated infections (HAIs) and the increasing number of antibiotic-resistant bacteria strains remain significant public health threats worldwide. Although the number of HAIs has decreased by using improved sterilization protocols, the cost related to HAIs is still quantified in billions of dollars. Furthermore, the development of multi-drug resistant strains is increasing exponentially, demonstrating that current treatments are inefficient. Thus, the quest for new methods to eradicate bacterial infection is increasingly important in antimicrobial, drug delivery and biomaterials research. Herein, the bactericidal activity of a water-soluble NO-releasing polysaccharide derivative was evaluated in nutrient broth media against three bacteria strains that are commonly responsible for HAIs. Data confirmed that this NO-releasing polysaccharide derivative induced an 8-log reduction in bacterial growth after 24h for Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus. Additionally, the absence of bacteria after 72 h of exposure to NO illustrates the inability of the bacteria to recover and the prevention of biofilm formation. The presented 8-log reduction in bacterial survival after 24h is among the highest reduction reported for NO delivery systems to date, and reaches the desired standard for industrially-relevant reduction. More specifically, this system represents the only water-soluble antimicrobial to reach such a significant bacterial reduction in nutrient rich media, wherein experimental conditions more closely mimic the in vivo environment than those in previous reports. Furthermore, the absence of bacterial activity after 72 h and the versatility of using a water-soluble compound suggest that this NO-releasing polysaccharide derivative is a promising route for treating HAIs.

Nitric oxide-releasing polysaccharide derivative exhibits 8-log reduction against Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus.

Health-care associated infections (HAIs) and the increasing number of antibiotic-resistant bacteria strains remain significant public health threats worldwide. Although the number of HAIs has decreased by using improved sterilization protocols, the cost related to HAIs is still quantified in billions of dollars. Furthermore, the development of multi-drug resistant strains is increasing exponentially, demonstrating that current treatments are inefficient. Thus, the quest for new methods to eradicate bacterial infection is increasingly important in antimicrobial, drug delivery and biomaterials research. Herein, the bactericidal activity of a water-soluble NO-releasing polysaccharide derivative was evaluated in nutrient broth media against three bacteria strains that are commonly responsible for HAIs. Data confirmed that this NO-releasing polysaccharide derivative induced an 8-log reduction in bacterial growth after 24h for Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus. Additionally, the absence of bacteria after 72h of exposure to NO illustrates the inability of the bacteria to recover and the prevention of biofilm formation. The presented 8-log reduction in bacterial survival after 24h is among the highest reduction reported for NO delivery systems to date, and reaches the desired standard for industrially-relevant reduction. More specifically, this system represents the only water-soluble antimicrobial to reach such a significant bacterial reduction in nutrient rich media, wherein experimental conditions more closely mimic the in vivo environment than those in previous reports. Furthermore, the absence of bacterial activity after 72h and the versatility of using a water-soluble compound suggest that this NO-releasing polysaccharide derivative is a promising route for treating HAIs.

Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response.

The YEATS domain, found in a number of chromatin-associated proteins, has recently been shown to have the capacity to bind histone lysine acetylation. Here, we show that the YEATS domain of Taf14, a member of key transcriptional and chromatin-modifying complexes in yeast, is a selective reader of histone H3 Lys9 acetylation (H3K9ac). Structural analysis reveals that acetylated Lys9 is sandwiched in an aromatic cage formed by F62 and W81. Disruption of this binding in cells impairs gene transcription and the DNA damage response. Our findings establish a highly conserved acetyllysine reader function for the YEATS domain protein family and highlight the significance of this interaction for Taf14.

Interrogating the function of metazoan histones using engineered gene clusters.

Histones and their posttranslational modifications influence the regulation of many DNA-dependent processes. Although an essential role for histone-modifying enzymes in these processes is well established, defining the specific contribution of individual histone residues remains a challenge because many histone-modifying enzymes have nonhistone targets. This challenge is exacerbated by the paucity of suitable approaches to genetically engineer histone genes in metazoans. Here, we describe a platform in Drosophila for generating and analyzing any desired histone genotype, and we use it to test the in vivo function of three histone residues. We demonstrate that H4K20 is neither essential for DNA replication nor for completion of development, unlike inferences drawn from analyses of H4K20 methyltransferases. We also show that H3K36 is required for viability and H3K27 is essential for maintenance of cellular identity but not for gene activation. These findings highlight the power of engineering histones to interrogate genome structure and function in animals.

A PWWP domain-containing protein targets the NuA3 acetyltransferase complex via histone H3 lysine 36 trimethylation to coordinate transcriptional elongation at coding regions.

Post-translational modifications of histones, such as acetylation and methylation, are differentially positioned in chromatin with respect to gene organization. For example, although histone H3 is often trimethylated on lysine 4 (H3K4me3) and acetylated on lysine 14 (H3K14ac) at active promoter regions, histone H3 lysine 36 trimethylation (H3K36me3) occurs throughout the open reading frames of transcriptionally active genes. The conserved yeast histone acetyltransferase complex, NuA3, specifically binds H3K4me3 through a plant homeodomain (PHD) finger in the Yng1 subunit, and subsequently catalyzes the acetylation of H3K14 through the histone acetyltransferase domain of Sas3, leading to transcription initiation at a subset of genes. We previously found that Ylr455w (Pdp3), an uncharacterized proline-tryptophan-tryptophan-proline (PWWP) domain-containing protein, copurifies with stable members of NuA3. Here, we employ mass-spectrometric analysis of affinity purified Pdp3, biophysical binding assays, and genetic analyses to classify NuA3 into two functionally distinct forms: NuA3a and NuA3b. Although NuA3a uses the PHD finger of Yng1 to interact with H3K4me3 at the 5'-end of open reading frames, NuA3b contains the unique member, Pdp3, which regulates an interaction between NuA3b and H3K36me3 at the transcribed regions of genes through its PWWP domain. We find that deletion of PDP3 decreases NuA3-directed transcription and results in growth defects when combined with transcription elongation mutants, suggesting NuA3b acts as a positive elongation factor. Finally, we determine that NuA3a, but not NuA3b, is synthetically lethal in combination with a deletion of the histone acetyltransferase GCN5, indicating NuA3b has a specialized role at coding regions that is independent of Gcn5 activity. Collectively, these studies define a new form of the NuA3 complex that associates with H3K36me3 to effect transcriptional elongation. MS data are available via ProteomeXchange with identifier PXD001156.

A sequence in the Drosophila H3-H4 Promoter triggers histone locus body assembly and biosynthesis of replication-coupled histone mRNAs.

Compartmentalization of RNA biosynthetic factors into nuclear bodies (NBs) is a ubiquitous feature of eukaryotic cells. How NBs initially assemble and ultimately affect gene expression remains unresolved. The histone locus body (HLB) contains factors necessary for replication-coupled histone messenger RNA transcription and processing and associates with histone gene clusters. Using a transgenic assay for ectopic Drosophila HLB assembly, we show that a sequence located between, and transcription from, the divergently transcribed H3-H4 genes nucleates HLB formation and activates other histone genes in the histone gene cluster. In the absence of transcription from the H3-H4 promoter, "proto-HLBs" (containing only a subset of HLB components) form, and the adjacent histone H2a-H2b genes are not expressed. Proto-HLBs also transiently form in mutant embryos with the histone locus deleted. We conclude that HLB assembly occurs through a stepwise process involving stochastic interactions of individual components that localize to a specific sequence in the H3-H4 promoter.