The NSL complex is required for piRNA production from telomeric clusters.

The NSL complex is a transcriptional activator. Germline-specific knockdown of NSL complex subunits NSL1, NSL2, and NSL3 results in reduced piRNA production from a subset of bidirectional piRNA clusters, accompanied by widespread transposon derepression. The piRNAs most transcriptionally affected by NSL2 and NSL1 RNAi map to telomeric piRNA clusters. At the chromatin level, these piRNA clusters also show decreased levels of H3K9me3, HP1a, and Rhino after NSL2 depletion. Using NSL2 ChIP-seq in ovaries, we found that this protein specifically binds promoters of telomeric transposons HeT-A, TAHRE, and TART Germline-specific depletion of NSL2 also led to a reduction in nuclear Piwi in nurse cells. Our findings thereby support a role for the NSL complex in promoting the transcription of piRNA precursors from telomeric piRNA clusters and in regulating Piwi levels in the Drosophila female germline.

Facultative dosage compensation of developmental genes on autosomes in Drosophila and mouse embryonic stem cells.

Haploinsufficiency and aneuploidy are two phenomena, where gene dosage alterations cause severe defects ultimately resulting in developmental failures and disease. One remarkable exception is the X chromosome, where copy number differences between sexes are buffered by dosage compensation systems. In Drosophila, the Male-Specific Lethal complex (MSLc) mediates upregulation of the single male X chromosome. The evolutionary origin and conservation of this process orchestrated by MSL2, the only male-specific protein within the fly MSLc, have remained unclear. Here, we report that MSL2, in addition to regulating the X chromosome, targets autosomal genes involved in patterning and morphogenesis. Precise regulation of these genes by MSL2 is required for proper development. This set of dosage-sensitive genes maintains such regulation during evolution, as MSL2 binds and similarly regulates mouse orthologues via Histone H4 lysine 16 acetylation. We propose that this gene-by-gene dosage compensation mechanism was co-opted during evolution for chromosome-wide regulation of the Drosophila male X.

A mutually exclusive stem-loop arrangement in roX2 RNA is essential for X-chromosome regulation in Drosophila.

The X chromosome provides an ideal model system to study the contribution of RNA-protein interactions in epigenetic regulation. In male flies, roX long noncoding RNAs (lncRNAs) harbor several redundant domains to interact with the ubiquitin ligase male-specific lethal 2 (MSL2) and the RNA helicase Maleless (MLE) for X-chromosomal regulation. However, how these interactions provide the mechanics of spreading remains unknown. By using the uvCLAP (UV cross-linking and affinity purification) methodology, which provides unprecedented information about RNA secondary structures in vivo, we identified the minimal functional unit of roX2 RNA. By using wild-type and various MLE mutant derivatives, including a catalytically inactive MLE derivative, MLEGET, we show that the minimal roX RNA contains two mutually exclusive stem-loops that exist in a peculiar structural arrangement: When one stem-loop is unwound by MLE, an alternate structure can form, likely trapping MLE in this perpetually structured region. We show that this functional unit is necessary for dosage compensation, as mutations that disrupt this formation lead to male lethality. Thus, we propose that roX2 lncRNA contains an MLE-dependent affinity switch to enable reversible interactions of the MSL complex to allow dosage compensation of the X chromosome.

An exogenous chloroplast genome for complex sequence manipulation in algae.

We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii. Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus. Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.

Long-term outcomes based on time-to-angioplasty in patients admitted with non-ST-segment elevation acute coronary syndromes.

OBJECTIVE: We investigated the impact of the duration from hospital admission to coronary angiography on the outcome of patients admitted with non ST-segment elevation acute coronary syndromes (NSTE-ACS). BACKGROUND: Invasive risk stratification in patients with acute coronary syndromes (ACS) has been shown to improve outcome in contemporary studies. It is unclear whether early coronary angiography is better than initial medical therapy with later angiography. METHODS: We performed an analysis of patients admitted to a tertiary coronary intensive care unit (CICU) with NSTE-ACS and had coronary angiography performed during the same hospitalization. Patients were categorized into three groups based on the time-to-angiography: same-day, 1 to 2 days, and > 2 days. The baseline clinical features, angiography results, 30-day, 6-month cardiovascular outcome and 3-year mortality rate were compared between the groups before and after adjusting for confounding variables. RESULTS: A total of 836 fulfilled the inclusion criteria. Patients undergoing angiography > 2 days had a higher incidence of 3-vessel disease (45.7% vs. 31.7%, p < 0.001), underwent less percutaneous interventions at the time of the angiography (41.6% vs. 56.7%, p < 0.001), and more frequent coronary artery bypass surgery (9.9% vs. 15.3%, p = 0.05). Patients undergoing late invasive risk stratification (> 2 days) had increased 3-year mortality (OR 2.12, 95% CI 1.03-4.35, p = 0.04) after adjusting for confounding variables. CONCLUSION: In patients with NSTE-ACS and no contraindication to angiography, delayed angiography of more than 2 days of presentation was associated with increased mortality at 3 years.