HP1 reshapes nucleosome core to promote phase separation of heterochromatin.

Heterochromatin affects genome function at many levels. It enables heritable gene repression, maintains chromosome integrity and provides mechanical rigidity to the nucleus1,2. These diverse functions are proposed to arise in part from compaction of the underlying chromatin2. A major type of heterochromatin contains at its core the complex formed between HP1 proteins and chromatin that is methylated on histone H3, lysine 9 (H3K9me). HP1 is proposed to use oligomerization to compact chromatin into phase-separated condensates3-6. Yet, how HP1-mediated phase separation relates to chromatin compaction remains unclear. Here we show that chromatin compaction by the Schizosaccharomyces pombe HP1 protein Swi6 results in phase-separated liquid condensates. Unexpectedly, we find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a nucleosome. Restraining these dynamics impairs compaction of chromatin into liquid droplets by Swi6. Our results indicate that Swi6 couples its oligomerization to the phase separation of chromatin by a counterintuitive mechanism, namely the dynamic exposure of buried nucleosomal regions. We propose that such reshaping of the octamer core by Swi6 increases opportunities for multivalent interactions between nucleosomes, thereby promoting phase separation. This mechanism may more generally drive chromatin organization beyond heterochromatin.

Discovery of a functionally selective ghrelin receptor (GHSR1a) ligand for modulating brain dopamine.

SignificanceThe modulation of growth hormone secretagogue receptor-1a (GHSR1a) signaling is a promising strategy for treating brain conditions of metabolism, aging, and addiction. GHSR1a activation results in pleiotropic physiological outcomes through distinct and pharmacologically separable G protein- and ß-arrestin (ßarr)-dependent signaling pathways. Thus, pathway-selective modulation can enable improved pharmacotherapeutics that can promote therapeutic efficacy while mitigating side effects. Here, we describe the discovery of a brain-penetrant small molecule, N8279 (NCATS-SM8864), that biases GHSR1a conformations toward Gαq activation and reduces aberrant dopaminergic behavior in mice. N8279 represents a promising chemical scaffold to advance the development of better treatments for GHSR1a-related brain disorders involving the pathological dysregulation of dopamine.

Reduction of GPSM3 expression akin to the arthritis-protective SNP rs204989 differentially affects migration in a neutrophil model.

G Protein Signaling Modulator-3 (GPSM3) is a leukocyte-specific regulator of G protein-coupled receptors (GPCRs), which binds inactivated Gαi·GDP subunits and precludes their reassociation with Gßγ subunits. GPSM3 deficiency protects mice from inflammatory arthritis and, in humans, GPSM3 single-nucleotide polymorphisms (SNPs) are inversely associated with the risk of rheumatoid arthritis development; recently, these polymorphisms were linked to one particular SNP (rs204989) that decreases GPSM3 transcript abundance. However, the precise role of GPSM3 in leukocyte biology is unknown. Here, we show that GPSM3 is induced in the human promyelocytic leukemia NB4 cell line following retinoic acid treatment, which differentiates this cell line into a model of neutrophil physiology (NB4*). Reducing GPSM3 expression in NB4* cells, akin to the effect ascribed to the rs204989 C>T transition, disrupts cellular migration toward leukotriene B4 (LTB4) and (to a lesser extent) interleukin-8 (a.k.a. IL-8 or CXCL8), but not migration toward formylated peptides (fMLP). As the chemoattractants LTB4 and CXCL8 are involved in recruitment of neutrophils to the arthritic joint, our results suggest that the arthritis-protective GPSM3 SNP rs204989 may act to decrease neutrophil chemoattractant responsiveness.

Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance.

G protein signaling modulator 3 (GPSM3) is a regulator of G protein-coupled receptor signaling, with expression restricted to leukocytes and lymphoid organs. Previous genome-wide association studies have highlighted single-nucleotide polymorphisms (SNPs; rs204989 and rs204991) in a region upstream of the GPSM3 transcription start site as being inversely correlated to the prevalence of rheumatoid arthritis (RA)-this association is supported by the protection afforded to Gpsm3-deficient mice in models of inflammatory arthritis. Here, we assessed the functional consequences of these polymorphisms. We collected biospecimens from 50 volunteers with RA diagnoses, 50 RA-free volunteers matched to the aforementioned group and 100 unmatched healthy young volunteers. We genotyped these individuals for GPSM3 (rs204989, rs204991), CCL21 (rs2812378) and HLA gene region (rs6457620) polymorphisms, and found no significant differences in minor allele frequencies between the RA and disease-free cohorts. However, we identified that individuals homozygous for SNPs rs204989 and rs204991 had decreased GPSM3 transcript abundance relative to individuals homozygous for the major allele. In vitro promoter activity studies suggest that SNP rs204989 is the primary cause of this decrease in transcript levels. Knockdown of GPSM3 in THP-1 cells, a human monocytic cell line, was found to disrupt ex vivo migration to the chemokine MCP-1.

A negative regulatory element in a prespore-specific promoter of dictyostelium discoideum(1).

We previously isolated several 'promoter-trap' transformants in which insertion of a promoterless beta-galactosidase gene into the genome caused expression of beta-galactosidase in specific cell types. The upstream flanking region was rescued from one transformant specifically expressing beta-galactosidase in prespore cells. We sequenced the promoter of the gene that is fused in-frame with lacZ and characterised a negative element that inhibits expression in pstO cells (a subtype of prestalk cells). Gel-retardation assays show that a developmentally regulated factor(s) recognises and binds to this element.

Characterisation of an intracellular Ca2+ pump in Dictyostelium.

Calcium uptake by microsomal membranes from the cellular slime mould Dictyostelium discoideum was measured using Calcium Green-2 as a fluorescent probe of external free Ca2+ concentration. High-affinity Ca2+ uptake was found to be completely inhibited by low concentrations of vanadate, but not by thapsigargin, suggesting that the activity is mediated by a Ca(2+)-ATPase distinct from sarco(endo)plasmic reticulum type of higher animal cells. On sucrose density gradients, Ca2+ uptake distributes with vacuolar proton pump activity and part of the observed Ca2+ uptake is dependent on the pH gradient generated by the vacuolar-type H(+)-ATPase, indicating that the Ca2+ pump is located on both acidic and non-acidic vesicles, possibly derived from the H(+)-ATPase-rich contractile vacuole complex.

Determination of the peptide torsion angle phi by 15N chemical shift and 13Calpha-1Halpha dipolar tensor correlation in solid-state MAS NMR.

We demonstrate a dipolar-chemical shift correlation technique for sign-sensitive determination of the torsion angle phi in solid peptides and proteins under magic-angle spinning. The indirect dimension of the experiment is obtained by separate but synchronous evolution of the magnetization under the 15N chemical shift and the C-H dipolar coupling. The resulting sum and difference spectrum of the two frequencies, with more than ten independent sidebands, depends strongly on the relative orientation of the 15N chemical shift tensor and the Calpha-Halpha bond. This relative orientation reflects the C(O)i-1-N-Calpha-C(O)i torsion angle. The technique can distinguish phi angles over the full range of 360 degrees when the amide 15N chemical shift tensor does not possess reflection symmetry with respect to the peptide plane. Thus it complements our previous HNCH experiment, in which two mirror-symmetric conformers of the HN-N bond relative to the Calpha-Halpha bond around the N-Calpha axis cannot be distinguished.

Coupling amplification in 2D MAS NMR and its application to torsion angle determination in peptides.

A technique for amplifying the apparent magnitudes of 13C-1H and 15N-1H dipolar interactions in magic-angle spinning experiments is described. By inserting rotor-synchronized 180 degrees pulses in the evolution period of a 2D dipolar-chemical shift experiment, heteronuclear dipolar couplings are doubled or quadrupled relative to the spinning speed. The increased number of dipolar sidebands is desirable for retaining structural information in the indirectly detected dipolar dimension while resolving inequivalent sites in the isotropic chemical shift dimension at relatively high spinning speeds. This coupling amplification method is incorporated into an experiment that determines the peptide torsion angle phi through the relative orientation of the Calpha-Halpha and N-HN bonds. It is shown both experimentally and theoretically that the angular resolution of the measurement is enhanced significantly by the selective doubling of the N-HN coupling.

Would you consider prescribing syringes to injection drug users? Addiction Medicine Conference Survey.

In order to assess attitudes and practices of physicians regarding prescribing syringes to injection drug users (IDUs) to prevent disease transmission, a survey was conducted at the 2000 ASAM Conference. Of 497 physicians, 104 responded, representing 30 states and 3 countries. Seventy-eight percent provided care for IDUs. Only 2% had prescribed syringes to IDUs for safer injection of illegal drugs. Nineteen percent had prescribed syringes to diabetic patients whom they believed would use the syringes for injecting illegal drugs. Overall, 61% of physicians (74% of internists, 37% of psychiatrists) (p = 0.04) would consider prescribing syringes to IDUs. Prescribing syringes to IDUs can be part of a comprehensive approach to preventing spread of HIV and other infections, decreasing complications of syringe reuse, and bringing IDUs into medical and substance abuse treatment. The majority of physicians surveyed expressed interest in prescribing syringes. Psychiatrists may be less willing to do so.

Developmental decisions in Dictyostelium discoideum.

A few hours after the onset of starvation, amoebae of Dictyostelium discoideum start to form multicellular aggregates by chemotaxis to centers that emit periodic cyclic AMP signals. There are two major developmental decisions: first, the aggregates either construct fruiting bodies directly, in a process known as culmination, or they migrate for a period as "slugs." Second, the amoebae differentiate into either prestalk or prespore cells. These are at first randomly distributed within aggregates and then sort out from each other to form polarized structures with the prestalk cells at the apex, before eventually maturing into the stalk cells and spores of fruiting bodies. Developmental gene expression seems to be driven primarily by cyclic AMP signaling between cells, and this review summarizes what is known of the cyclic AMP-based signaling mechanism and of the signal transduction pathways leading from cell surface cyclic AMP receptors to gene expression. Current understanding of the factors controlling the two major developmental choices is emphasized. The weak base ammonia appears to play a key role in preventing culmination by inhibiting activation of cyclic AMP-dependent protein kinase, whereas the prestalk cell-inducing factor DIF-1 is central to the choice of cell differentiation pathway. The mode of action of DIF-1 and of ammonia in the developmental choices is discussed.

The effect of ammonia on cell-type-specific enzyme accumulation in Dictyostelium discoideum.

We have shown that ammonia inhibits stalk cell formation and promotes spore formation during cyclic AMP induced differentiation of monolayers of sporogenous amoebae (Gross, J.D. et al., Nature (London), 303, 244-245, 1983). Here we show that exposure to ammonia favours the accumulation of prespore- over prestalk-specific enzymes and related products. We conclude that ammonia switches cells from the prestalk to the prespore pathway rather than simply inhibiting stalk cell maturation and hence may act as a morphogen during development.

Anion effects on vesicle acidification in Dictyostelium.

Chloride ions stimulated the ATP-dependent formation of a proton gradient in vesicles derived from amoebae of the cellular slime mould, D. discoideum, and reduced the formation of a membrane potential, inhibited rather than stimulated the formation of the proton gradient. Since bicarbonate ions did not inhibit H(+)-ATPase activity we conclude that they enter the vesicles and combine with translocated protons. This finding is consistent with the suggestion that the membranes of the light vesicle fraction are fragments of contractile vacuole complexes, and that these organelles increase their osmotic activity by taking up bicarbonate ions and protons from the cytoplasm, and then release water and carbonic acid into the extracellular milieu.