Replication stress affects the fidelity of nucleosome-mediated epigenetic inheritance.

The fidelity of epigenetic inheritance or, the precision by which epigenetic information is passed along, is an essential parameter for measuring the effectiveness of the process. How the precision of the process is achieved or modulated, however, remains largely elusive. We have performed quantitative measurement of epigenetic fidelity, using position effect variegation (PEV) in Schizosaccharomyces pombe as readout, to explore whether replication perturbation affects nucleosome-mediated epigenetic inheritance. We show that replication stresses, due to either hydroxyurea treatment or various forms of genetic lesions of the replication machinery, reduce the inheritance accuracy of CENP-A/Cnp1 nucleosome positioning within centromere. Mechanistically, we demonstrate that excessive formation of single-stranded DNA, a common molecular abnormality under these conditions, might have correlation with the reduction in fidelity of centromeric chromatin duplication. Furthermore, we show that replication stress broadly changes chromatin structure at various loci in the genome, such as telomere heterochromatin expanding and mating type locus heterochromatin spreading out of the boundaries. Interestingly, the levels of inheritable expanding at sub-telomeric heterochromatin regions are highly variable among independent cell populations. Finally, we show that HU treatment of the multi-cellular organisms C. elegans and D. melanogaster affects epigenetically programmed development and PEV, illustrating the evolutionary conservation of the phenomenon. Replication stress, in addition to its demonstrated role in genetic instability, promotes variable epigenetic instability throughout the epigenome.

Operating Modes and Cooling Capabilities of the 3-Stage ADR Developed for the Soft-X-ray Spectrometer Instrument on Astro-H.

A 3-stage adiabatic demagnetization refrigerator (ADR)[1] is used on the Soft X-ray Spectrometer instrument[2] on Astro-H[3] to cool a 6×6 array of x-ray microcalorimeters to 50 mK. The ADR is supported by a cryogenic system[4] consisting of a superfluid helium tank, a 4.5 K Joule-Thomson (JT) cryocooler, and additional 2-stage Stirling cryocoolers that pre-cool the JT cooler and cool radiation shields within the cryostat. The ADR is configured so that it can use either the liquid helium or the JT cryocooler as its heat sink, giving the instrument an unusual degree of tolerance for component failures or degradation in the cryogenic system. The flight detector assembly, ADR and dewar were integrated into the flight dewar in early 2014, and have since been extensively characterized and calibrated. This paper summarizes the operation and performance of the ADR in all of its operating modes.

Autoregulation of the Drosophila Noncoding roX1 RNA Gene.

Most genes along the male single X chromosome in Drosophila are hypertranscribed about two-fold relative to each of the two female X chromosomes. This is accomplished by the MSL (male-specific lethal) complex that acetylates histone H4 at lysine 16. The MSL complex contains two large noncoding RNAs, roX1 (RNA on X) and roX2, that help target chromatin modifying enzymes to the X. The roX RNAs are functionally redundant but differ in size, sequence, and transcriptional control. We wanted to find out how roX1 production is regulated. Ectopic DC can be induced in wild-type (roX1(+) roX2(+)) females if we provide a heterologous source of MSL2. However, in the absence of roX2, we found that roX1 expression failed to come on reliably. Using an in situ hybridization probe that is specific only to endogenous roX1, we found that expression was restored if we introduced either roX2 or a truncated but functional version of roX1. This shows that pre-existing roX RNA is required to positively autoregulate roX1 expression. We also observed massive cis spreading of the MSL complex from the site of roX1 transcription at its endogenous location on the X chromosome. We propose that retention of newly assembled MSL complex around the roX gene is needed to drive sustained transcription and that spreading into flanking chromatin contributes to the X chromosome targeting specificity. Finally, we found that the gene encoding the key male-limited protein subunit, msl2, is transcribed predominantly during DNA replication. This suggests that new MSL complex is made as the chromatin template doubles. We offer a model describing how the production of roX1 and msl2, two key components of the MSL complex, are coordinated to meet the dosage compensation demands of the male cell.

Mutations in the transcription elongation factor SPT5 disrupt a reporter for dosage compensation in Drosophila.

In Drosophila, the MSL (Male Specific Lethal) complex up regulates transcription of active genes on the single male X-chromosome to equalize gene expression between sexes. One model argues that the MSL complex acts upon the elongation step of transcription rather than initiation. In an unbiased forward genetic screen for new factors required for dosage compensation, we found that mutations in the universally conserved transcription elongation factor Spt5 lower MSL complex dependent expression from the miniwhite reporter gene in vivo. We show that SPT5 interacts directly with MSL1 in vitro and is required downstream of MSL complex recruitment, providing the first mechanistic data corroborating the elongation model of dosage compensation.

A new strategy for isolating genes controlling dosage compensation in Drosophila using a simple epigenetic mosaic eye phenotype.

BACKGROUND: The Drosophila Male Specific Lethal (MSL) complex contains chromatin modifying enzymes and non-coding roX RNA. It paints the male X at hundreds of bands where it acetylates histone H4 at lysine 16. This epigenetic mark increases expression from the single male X chromosome approximately twofold above what gene-specific factors produce from each female X chromosome. This equalises X-linked gene expression between the sexes. Previous screens for components of dosage compensation relied on a distinctive male-specific lethal phenotype. RESULTS: Here, we report a new strategy relying upon an unusual male-specific mosaic eye pigmentation phenotype produced when the MSL complex acts upon autosomal roX1 transgenes. Screening the second chromosome identified at least five loci, two of which are previously described components of the MSL complex. We focused our analysis on the modifier alleles of MSL1 and MLE (for 'maleless'). The MSL1 lesions are not simple nulls, but rather alter the PEHE domain that recruits the MSL3 chromodomain and MOF ('males absent on first') histone acetyltransferase subunits to the complex. These mutants are compromised in their ability to recruit MSL3 and MOF, dosage compensate the X, and support long distance spreading from roX1 transgenes. Yet, paradoxically, they were isolated because they somehow increase MSL complex activity immediately around roX1 transgenes in combination with wild-type MSL1 subunits. CONCLUSIONS: We propose that these diverse phenotypes arise from perturbations in assembly of MSL subunits onto nascent roX transcripts. This strategy is a promising alternative route for identifying previously unknown components of the dosage compensation pathway and novel alleles of known MSL proteins.

Precision measurement of the K-shell spectrum from highly charged xenon with an array of X-ray calorimeters.

We present a measurement of the K-shell spectrum from highly charged xenon ions recorded with a high-energy x-ray calorimeter spectrometer array that can distinguish between various theories for the atomic structure of the two electron system. The array was designed to provide high resolution with high quantum efficiency in the 10-60 keV x-ray range which allows us to resolve blends that afflicted previous measurements. A precision of better than 2 eV was achieved in the measurement of the Xe52+ and Xe53+ K-shell transitions located near 31 keV, which is an order of magnitude better than previously reported.

The Drosophila roX1 RNA gene can overcome silent chromatin by recruiting the male-specific lethal dosage compensation complex.

The Drosophila MSL complex consists of at least six proteins and two noncoding roX RNAs that mediate dosage compensation. It acts to remodel the male's X chromatin by covalently modifying the amino terminal tails of histones. The roX1 and roX2 genes are thought to be nucleation sites for assembly and spreading of MSL complexes into surrounding chromatin where they roughly double the rates of transcription. We generated many transgenic stocks in which the roX1 gene was moved from its normal location on the X to new autosomal sites. Approximately 10% of such lines displayed unusual sexually dimorphic expression patterns of the transgene's mini-white eye-color marker. Males often displayed striking mosaic pigmentation patterns similar to those seen in position-effect variegation and yet most inserts were in euchromatic locations. In many of these stocks, female mini-white expression was very low or absent. The male-specific activation of mini-white depended upon the MSL complex. We propose that these transgenes are inserted in several different types of repressive chromatin environments that inhibit mini-white expression. Males are able to overcome this silencing through the action of the MSL complex spreading from the roX1 gene and remodeling the local chromatin to allow transcription. The potency with which an ectopic MSL complex overcomes silent chromatin suggests that its normal action on the X must be under strict regulation.

The Drosophila over compensating males gene genetically inhibits dosage compensation in males.

Male Drosophila are monosomic for the X chromosome, but survive due to dosage compensation. They use the Male Specific Lethal (MSL) complex composed of noncoding roX RNA and histone modifying enzymes to hypertranscribe most genes along the X ∼1.6-1.8 fold relative to each female allele. It is not known how the MSL complex achieves this precise adjustment to a large and diverse set of target genes. We carried out a genetic screen searching for novel factors that regulate dosage compensation in flies. This strategy generated thirty alleles in a previously uncharacterized gene, over compensating males (ocm) that antagonizes some aspect of MSL activity. The mutations were initially recovered because they derepressed an MSL-dependent eye color reporter. Null ocm mutations are lethal to both sexes early in development revealing an essential function. Combinations of hypomorphic ocm alleles display a male specific lethality similar to mutations in the classic msl genes, but ocm males die due to excessive, rather than lack of dosage compensation. Males that die due to very low MSL activity can be partially rescued by ocm mutations. Likewise, males that would die from ocm mutations can be rescued by reducing the dose of various msl and roX genes. ocm encodes a large nuclear protein that shares a novel cysteine rich motif with known transcription factors.

Transcription rate of noncoding roX1 RNA controls local spreading of the Drosophila MSL chromatin remodeling complex.

The dosage compensation complex in Drosophila is composed of at least five MSL proteins and two noncoding roX RNAs that bind hundreds of sites along the single male X chromosome. The roX RNAs are transcribed from X-linked genes and their RNA products "paint" the male X. The roX RNAs and bound MSL proteins can spread in cis from sites of roX transcription, but the mechanism controlling spreading is unknown. Here we find that cis spreading from autosomal roX1 transgenes is coupled to the level of roX transcription. Low to moderate transcription favors, and vigorous transcription abolishes local spreading. We constructed a roX1 minigene one third the size of wild type as a starting point for mutagenesis. This allowed us to test which evolutionarily conserved motifs were required for activity. One short repeat element shared between roX1 and roX2 was found to be particularly important. When all copies were deleted, the RNA was inactive and unstable, while extra copies seem to promote local spreading of the MSL complex from sites of roX1 synthesis. We propose that assembly of the MSL proteins onto the extreme 3' region of elongating roX1 transcripts determines whether the MSL complex spreads in cis.

High-resolution hard x-ray spectroscopy of high-temperature plasmas using an array of quantum microcalorimeters.

Quantum microcalorimeters show promise in being able to fully resolve x-ray spectra from heavy highly charged ions, such as would be found in hot plasmas with temperatures in excess of 50 keV. Quantum microcalorimeter arrays are able to achieve this as they have a high-resolving power and good effective quantum efficiency for hard x-ray photons up to 60 keV. To demonstrate this, we present a measurement using an array of thin HgTe quantum microcalorimeters to measure the K-shell spectrum of hydrogenlike through carbonlike praseodymium (Z=57). With this device we are able to attain a resolving power, E/DeltaE, of 1000 at a photon energy of 37 keV.

Performance of the EBIT calorimeter spectrometer.

The EBIT calorimeter spectrometer (ECS) is a new high-resolution, broadband x-ray spectrometer that has recently been installed at the Electron Beam Ion Trap Facility (EBIT) at the Lawrence Livermore National Laboratory. The ECS is an entirely new production class spectrometer that replaces the XRS/EBIT spectrometer that has been operating at EBIT since 2000. The ECS utilizes a 32-pixel x-ray calorimeter array from the XRS instrument on the Suzaku x-ray observatory. Eighteen of the pixels are optimized for the 0.1-10 keV band and yield 4.5 eV full width at half maximum energy resolution and 95% quantum efficiency at 6 keV. In addition, the ECS includes 14 detector pixels that are optimized for the high-energy band with a bandpass from 0.5 to over 100 keV with 34 eV resolution and 32% quantum efficiency at 60 keV. The ECS detector array is operated at 50 mK using a five stage cryogenic system that is entirely automated. The instrument takes data continuously for over 65 h with a 2.5 h recycle time. The ECS is a nondispersive, broadband, highly efficient spectrometer that is one of the prime instruments at the EBIT facility. The instrument is used for studies of absolute cross sections, charge exchange recombination, and x-ray emission from nonequilibrium plasmas, among other measurements in our laboratory astrophysics program.

Path to equality strewn with roX.

Male flies hypertranscribe most genes along their single X chromosome to match the output of females with two X chromosomes. This is mediated by chromatin modifications carried out by the MSL complex composed of noncoding roX RNA and at least five MSL proteins. New results indicate that one of these subunits, the MOF acetyltransferase, not only acts on histone H4, but on itself and MSL3. Cycles of covalent modifications of the MSL subunits may determine the proper level of hypertranscription or control cis spreading along the chromosome. The MSL complex binds to the roX genes, the very source of the RNA component of the complex. New details of how this interaction occurs hint at a possible autoregulatory function. Finally, despite intensive efforts, the molecular mechanism by which the MSL complex distinguishes the X from the autosomes remains a mystery. The MSL complex is able to spread epigenetically from the site of roX transcription, and recent work has defined the conditions that control local cis spreading. However, it is equally clear that soluble MSL complex can distinguish the X chromosome from autosomes. Reconciling all these findings into a unified model presents a challenge.

Extent of chromatin spreading determined by roX RNA recruitment of MSL proteins.

The untranslated roX1 and roX2 RNAs are components of the Drosophila male-specific lethal (MSL) complex, which modifies histones to up-regulate transcription of the male X chromosome. roX genes are normally located on the X chromosome, and roX transgenes can misdirect the dosage compensation machinery to spread locally on other chromosomes. Here we define MSL protein abundance as a determinant of whether the MSL complex will spread in cis from an autosomal roX transgene. The number of expressed roX genes in a nucleus was inversely correlated with spreading from roX transgenes. We suggest a model in which MSL proteins assemble into active complexes by binding nascent roX transcripts. When MSL protein/roX RNA ratios are high, assembly will be efficient, and complexes may be completed while still tethered to the DNA template. We propose that this local production of MSL complexes determines the extent of spreading into flanking chromatin.