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

Aerodynamic Analysis of Simulated Heat Shield Recession for the Orion Command Module

The aerodynamic effects of the recession of the ablative thermal protection system for the Orion Command Module of the Crew Exploration Vehicle are important for the vehicle guidance. At the present time, the aerodynamic effects of recession being handled within the Orion aerodynamic database indirectly with an additional safety factor placed on the uncertainty bounds. This study is an initial attempt to quantify the effects for a particular set of recessed geometry shapes, in order to provide more rigorous analysis for managing recession effects within the aerodynamic database. The aerodynamic forces and moments for the baseline and recessed geometries were computed at several trajectory points using multiple CFD codes, both viscous and inviscid. The resulting aerodynamics for the baseline and recessed geometries were compared. The forces (lift, drag) show negligible differences between baseline and recessed geometries. Generally, the moments show a difference between baseline and recessed geometries that correlates with the maximum amount of recession of the geometry. The difference between the pitching moments for the baseline and recessed geometries increases as Mach number decreases (and the recession is greater), and reach a value of -0.0026 for the lowest Mach number. The change in trim angle of attack increases from approx. 0.5deg at M = 28.7 to approx. 1.3deg at M = 6, and is consistent with a previous analysis with a lower fidelity engineering tool. This correlation of the present results with the engineering tool results supports the continued use of the engineering tool for future work. The present analysis suggests there does not need to be an uncertainty due to recession in the Orion aerodynamic database for the force quantities. The magnitude of the change in pitching moment due to recession is large enough to warrant inclusion in the aerodynamic database. An increment in the uncertainty for pitching moment could be calculated from these results and included in the development of the aerodynamic database uncertainty for pitching moment

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

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

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

Assessing karyotype precision by microarray-based comparative genomic hybridization in the myelodysplastic/myeloproliferative syndromes

<p>Abstract</p> <p>Background</p> <p>Recent genome-wide microarray-based research investigations have revealed a high frequency of submicroscopic copy number alterations (CNAs) in the myelodysplastic syndromes (MDS), suggesting microarray-based comparative genomic hybridization (aCGH) has the potential to detect new clinically relevant genomic markers in a diagnostic laboratory.</p> <p>Results</p> <p>We performed an exploratory study on 30 cases of MDS, myeloproliferative neoplasia (MPN) or evolving acute myeloid leukemia (AML) (% bone marrow blasts ≤ 30%, range 0-30%, median, 8%) by aCGH, using a genome-wide bacterial artificial chromosome (BAC) microarray. The sample data were compared to corresponding cytogenetics, fluorescence <it>in situ </it>hybridization (FISH), and clinical-pathological findings. Previously unidentified imbalances, in particular those considered submicroscopic aberrations (< 10 Mb), were confirmed by FISH analysis. CNAs identified by aCGH were concordant with the cytogenetic/FISH results in 25/30 (83%) of the samples tested. aCGH revealed new CNAs in 14/30 (47%) patients, including 28 submicroscopic or hidden aberrations verified by FISH studies. Cryptic 344-kb <it>RUNX1 </it>deletions were found in three patients at time of AML transformation. Other hidden CNAs involved 3q26.2/EVI1, 5q22/APC, 5q32/TCERG1,12p13.1/EMP1, 12q21.3/KITLG, and 17q11.2/NF1. Gains of CCND2/12p13.32 were detected in two patients. aCGH failed to detect a balanced translocation (n = 1) and low-level clonality (n = 4) in five karyotypically aberrant samples, revealing clinically important assay limitations.</p> <p>Conclusions</p> <p>The detection of previously known and unknown genomic alterations suggests that aCGH has considerable promise for identification of both recurring microscopic and submicroscopic genomic imbalances that contribute to myeloid disease pathogenesis and progression. These findings suggest that development of higher-resolution microarray platforms could improve karyotyping in clinical practice.</p

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 mRNA 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

Interrogating the Function of Metazoan Histones using Engineered Gene Clusters

Histones and their post-translational 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 non-histone targets. This challenge is exacerbated by the paucity of suitable approaches to genetically engineer histone genes in metazoans. Here, we describe a facile 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 conclusions drawn from analyses of H4K20 methyltransferases. We also show that H3K36 is required for viability and H3K27 is essential for maintenance of cellular identity during development. These findings highlight the power of engineering histones to interrogate genome structure and function in animals