Phosphorylation-Mediated Control of Histone Chaperone ASF1 Levels by Tousled-Like Kinases

Histone chaperones are at the hub of a diverse interaction networks integrating a plethora of chromatin modifying activities. Histone H3/H4 chaperone ASF1 is a target for cell-cycle regulated Tousled-like kinases (TLKs) and both proteins cooperate during chromatin replication. However, the precise role of post-translational modification of ASF1 remained unclear. Here, we identify the TLK phosphorylation sites for both Drosophila and human ASF1 proteins. Loss of TLK-mediated phosphorylation triggers hASF1a and dASF1 degradation by proteasome-dependent and independent mechanisms respectively. Consistent with this notion, introduction of phosphorylation-mimicking mutants inhibits hASF1a and dASF1 degradation. Human hASF1b is also targeted for proteasome-dependent degradation, but its stability is not affected by phosphorylation indicating that other mechanisms are likely to be involved in control of hASF1b levels. Together, these results suggest that ASF1 cellular levels are tightly controlled by distinct pathways and provide a molecular mechanism for post-translational regulation of dASF1 and hASF1a by TLK kinases

Late Replication Domains in Polytene and Non-Polytene Cells of Drosophila melanogaster

In D. melanogaster polytene chromosomes, intercalary heterochromatin (IH) appears as large dense bands scattered in euchromatin and comprises clusters of repressed genes. IH displays distinctly low gene density, indicative of their particular regulation. Genes embedded in IH replicate late in the S phase and become underreplicated. We asked whether localization and organization of these late-replicating domains is conserved in a distinct cell type. Using published comprehensive genome-wide chromatin annotation datasets (modENCODE and others), we compared IH organization in salivary gland cells and in a Kc cell line. We first established the borders of 60 IH regions on a molecular map, these regions containing underreplicated material and encompassing ∼12% of Drosophila genome. We showed that in Kc cells repressed chromatin constituted 97% of the sequences that corresponded to IH bands. This chromatin is depleted for ORC-2 binding and largely replicates late. Differences in replication timing between the cell types analyzed are local and affect only sub-regions but never whole IH bands. As a rule such differentially replicating sub-regions display open chromatin organization, which apparently results from cell-type specific gene expression of underlying genes. We conclude that repressed chromatin organization of IH is generally conserved in polytene and non-polytene cells. Yet, IH domains do not function as transcription- and replication-regulatory units, because differences in transcription and replication between cell types are not domain-wide, rather they are restricted to small “islands” embedded in these domains. IH regions can thus be defined as a special class of domains with low gene density, which have narrow temporal expression patterns, and so displaying relatively conserved organization

Repressive LTR Nucleosome Positioning by the BAF Complex Is Required for HIV Latency

The SWI/SNF BAF chromatin remodeling complex generates a repressive nucleosome structure at the HIV LTR conducive to establishment and maintenance of HIV latency, while PBAF augments HIV transcription

Essential Functions of the Histone Demethylase Lid

Drosophila Little imaginal discs (Lid) is a recently described member of the JmjC domain class of histone demethylases that specifically targets trimethylated histone H3 lysine 4 (H3K4me3). To understand its biological function, we have utilized a series of Lid deletions and point mutations to assess the role that each domain plays in histone demethylation, in animal viability, and in cell growth mediated by the transcription factor dMyc. Strikingly, we find that lid mutants are rescued to adulthood by either wildtype or enzymatically inactive Lid expressed under the control of its endogenous promoter, demonstrating that Lid's demethylase activity is not essential for development. In contrast, ubiquitous expression of UAS-Lid transgenes lacking its JmjN, C-terminal PHD domain, and C5HC2 zinc finger were unable to rescue lid homozygous mutants, indicating that these domains carry out Lid's essential developmental functions. Although Lid-dependent demethylase activity is not essential, dynamic removal of H3K4me3 may still be an important component of development, as we have observed a genetic interaction between lid and another H3K4me3 demethylase, dKDM2. We also show that Lid's essential C-terminal PHD finger binds specifically to di- and trimethylated H3K4 and that this activity is required for Lid to function in dMyc-induced cell growth. Taken together, our findings highlight the importance of Lid function in the regulated removal and recognition of H3K4me3 during development

Inclusive V0V^0 Production Cross Sections from 920 GeV Fixed Target Proton-Nucleus Collisions

Inclusive differential cross sections $d\sigma_{pA}/dx_F$ and $d\sigma_{pA}/dp_t^2$ for the production of \kzeros, \lambdazero, and \antilambda particles are measured at HERA in proton-induced reactions on C, Al, Ti, and W targets. The incident beam energy is 920 GeV, corresponding to $\sqrt {s} = 41.6$ GeV in the proton-nucleon system. The ratios of differential cross sections \rklpa and \rllpa are measured to be $6.2\pm 0.5$ and $0.66\pm 0.07$, respectively, for \xf $\approx-0.06$. No significant dependence upon the target material is observed. Within errors, the slopes of the transverse momentum distributions $d\sigma_{pA}/dp_t^2$ also show no significant dependence upon the target material. The dependence of the extrapolated total cross sections $\sigma_{pA}$ on the atomic mass $A$ of the target material is discussed, and the deduced cross sections per nucleon $\sigma_{pN}$ are compared with results obtained at other energies.Comment: 17 pages, 7 figures, 5 table

SWI/SNF and Asf1 Independently Promote Derepression of the DNA Damage Response Genes under Conditions of Replication Stress

The histone chaperone Asf1 and the chromatin remodeler SWI/SNF have been separately implicated in derepression of the DNA damage response (DDR) genes in yeast cells treated with genotoxins that cause replication interference. Using genetic and biochemical approaches, we have tested if derepression of the DDR genes in budding yeast involves functional interplay between Asf1 and SWI/SNF. We find that Asf1 and SWI/SNF are both recruited to DDR genes under replication stress triggered by hydroxyurea, and have detected a soluble complex that contains Asf1 and the Snf2 subunit of SWI/SNF. SWI/SNF recruitment to DDR genes however does not require Asf1, and deletion of Snf2 does not affect Asf1 occupancy of DDR gene promoters. A checkpoint engagement defect is sufficient to explain the synthetic effect of deletion of ASF1 and SNF2 on derepression of the DDR genes in hydroxyurea-treated cells. Collectively, our results show that the DDR genes fall into a class in which Asf1 and SWI/SNF independently control transcriptional induction

Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodelling

Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I

Perturbation Analysis of Heterochromatin-Mediated Gene Silencing and Somatic Inheritance

Repetitive sequences in eukaryotic genomes induce chromatin-mediated gene-silencing of juxtaposed genes. Many components that promote or antagonize silencing have been identified, but how heterochromatin causes variegated and heritable changes in gene expression remains mysterious. We have used inducible mis-expression in the Drosophila eye to recover new factors that alter silencing caused by the bwD allele, an insertion of repetitive satellite DNA that silences a bw+ allele on the homologous chromosome. Inducible modifiers allow perturbation of silencing at different times in development, and distinguish factors that affect establishment or maintenance of silencing. We find that diverse chromatin and RNA processing factors can de-repress silencing. Most factors are effective even in differentiated cells, implying that silent chromatin remains plastic. However, over-expression of the bantam microRNA or the crooked-legs (crol) zinc-finger protein only de-repress silencing when expressed in cycling cells. Over-expression of crol accelerates the cell cycle, and this is required for de-repression of silencing. Strikingly, continual over-expression of crol converts the speckled variegation pattern of bwD into sectored variegation, where de-repression is stably inherited through mitotic divisions. Over-expression of crol establishes an open chromatin state, but the factor is not needed to maintain this state. Our analysis reveals that active chromatin states can be efficiently inherited through cell divisions, with implications for the stable maintenance of gene expression patterns through development

Blood transcriptomics of drug-na\uefve sporadic Parkinson's disease patients

BACKGROUND: Parkinson's disease (PD) is a chronic progressive neurodegenerative disorder that is clinically defined in terms of motor symptoms. These are preceded by prodromal non-motor manifestations that prove the systemic nature of the disease. Identifying genes and pathways altered in living patients provide new information on the diagnosis and pathogenesis of sporadic PD. METHODS: Changes in gene expression in the blood of 40 sporadic PD patients and 20 healthy controls ("Discovery set") were analyzed by taking advantage of the Affymetrix platform. Patients were at the onset of motor symptoms and before initiating any pharmacological treatment. Data analysis was performed by applying Ranking-Principal Component Analysis, PUMA and Significance Analysis of Microarrays. Functional annotations were assigned using GO, DAVID, GSEA to unveil significant enriched biological processes in the differentially expressed genes. The expressions of selected genes were validated using RT-qPCR and samples from an independent cohort of 12 patients and controls ("Validation set"). RESULTS: Gene expression profiling of blood samples discriminates PD patients from healthy controls and identifies differentially expressed genes in blood. The majority of these are also present in dopaminergic neurons of the Substantia Nigra, the key site of neurodegeneration. Together with neuronal apoptosis, lymphocyte activation and mitochondrial dysfunction, already found in previous analysis of PD blood and post-mortem brains, we unveiled transcriptome changes enriched in biological terms related to epigenetic modifications including chromatin remodeling and methylation. Candidate transcripts as CBX5, TCF3, MAN1C1 and DOCK10 were validated by RT-qPCR. CONCLUSIONS: Our data support the use of blood transcriptomics to study neurodegenerative diseases. It identifies changes in crucial components of chromatin remodeling and methylation machineries as early events in sporadic PD suggesting epigenetics as target for therapeutic intervention

IL-10 transcription is negatively regulated by BAF180, a component of the SWI/SNF chromatin remodeling enzyme

<p>Abstract</p> <p>Background</p> <p>SWI/SNF chromatin remodeling enzymes play a critical role in the development of T helper lymphocytes, including Th2 cells, and directly program chromatin structure at Th2 cytokine genes. Different versions of SWI/SNF complexes, including BAF and PBAF, have been described based on unique subunit composition. However, the relative role of BAF and PBAF in Th cell function and cytokine expression has not been reported.</p> <p>Results</p> <p>Here we examine the role of the PBAF SWI/SNF complex in Th cell development and gene expression using mice deficient for a PBAF-specific component, BAF180. We find that T cell development in the thymus and lymphoid periphery is largely normal when the BAF180 gene is deleted late in thymic development. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus.</p> <p>Conclusions</p> <p>These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription.</p