A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation.

Organismal development and cell differentiation critically depend on chromatin state transitions. However, certain developmentally regulated genes lack histone 3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac, respectively) and histone 3 lysine 4 (H3K4) methylation, histone modifications common to most active genes. Here we describe a chromatin state featuring unique histone 3 lysine 14 acetylation (H3K14ac) peaks in key tissue-specific genes in Drosophila and human cells. Replacing H3K14 in Drosophila demonstrates that H3K14 is essential for expression of genes devoid of canonical histone modifications in the embryonic gut and larval wing imaginal disc, causing lethality and defective wing patterning. We find that the SWI/SNF protein Brahma (Brm) recognizes H3K14ac, that brm acts in the same genetic pathway as H3K14R, and that chromatin accessibility at H3K14ac-unique genes is decreased in H3K14R mutants. Our results show that acetylation of a single lysine is essential at genes devoid of canonical histone marks and uncover an important requirement for H3K14 in tissue-specific gene regulation.

Bundling Colorectal Cancer Screening Outreach with Screening for Social Risk in Federally Qualified Health Centers: A Stepped-Wedge Implementation-Effectiveness Study.

BACKGROUND: Bundling is combining individual interventions to meet quality metrics. Bundling offers of cancer screening with screening for social determinants of health (SDOH) may enable health centers to assist patients with social risks and yield efficiencies. OBJECTIVE: To measure effects of bundling fecal immunochemical testing (FIT) and SDOH screening in federally qualified health centers (FQHCs). DESIGN: Clustered stepped-wedge trial. PARTICIPANTS: Four Massachusetts FQHCs randomized to implement bundled FIT-SDOH over 8-week "steps." INTERVENTION: Outreach to 50-75-year-olds overdue for CRC screening to offer FIT with SDOH screening. The implementation strategy used facilitation and training for data monitoring and reporting. MAIN MEASURES: Implementation process descriptions, data from facilitation meetings, and CRC and SDOH screening rates. Rates were compared between implementation and control FQHCs in each "step" by fitting generalized linear mixed-effects models with random intercepts for FQHCs, patients, and "step" by FQHC. KEY RESULTS: FQHCs tailored implementation processes to their infrastructure, workflows, and staffing and prioritized different groups for outreach. Two FQHCs used population health outreach, and two integrated FIT-SDOH within established programs, such as pre-visit planning. Of 34,588 patients overdue for CRC screening, 54% were female; 20% Black, 11% Latino, 10% Asian, and 47% white; 32% had Medicaid, 16% Medicare, 32% private insurance, and 11% uninsured. Odds of CRC screening completion in implementation "steps" compared to controls were higher overall and among groups prioritized for outreach (overall: adjusted odds ratio (aOR) 2.41, p = 0.005; prioritized: aOR 2.88, p = 0.002). Odds of SDOH screening did not differ across "steps." CONCLUSIONS: As healthcare systems are required to conduct more screenings, it is notable that outreach for a long-standing cancer screening requirement increased screening, even when bundled with a newer screening requirement. This outreach was feasible in a real-world safety-net clinical population and may conserve resources, especially compared to more complex or intensive outreach strategies. CLINICAL TRIALS REGISTRATION: NCT04585919.

Spatiotemporally controlled generation of NTPs for single-molecule studies.

Many essential processes in the cell depend on proteins that use nucleoside triphosphates (NTPs). Methods that directly monitor the often-complex dynamics of these proteins at the single-molecule level have helped to uncover their mechanisms of action. However, the measurement throughput is typically limited for NTP-utilizing reactions, and the quantitative dissection of complex dynamics over multiple sequential turnovers remains challenging. Here we present a method for controlling NTP-driven reactions in single-molecule experiments via the local generation of NTPs (LAGOON) that markedly increases the measurement throughput and enables single-turnover observations. We demonstrate the effectiveness of LAGOON in single-molecule fluorescence and force spectroscopy assays by monitoring DNA unwinding, nucleosome sliding and RNA polymerase elongation. LAGOON can be readily integrated with many single-molecule techniques, and we anticipate that it will facilitate studies of a wide range of crucial NTP-driven processes.

Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity Switch Protein in the Yersinia Type III Secretion System.

Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia, the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy. The binding interface is centered on the C-terminal domain of YscU. Disrupting the YscU-YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the binding of YscP to the slowly self-cleaving YscU variant P264A conferred significant protection against autoproteolysis. The YscP-mediated inhibition of YscU autoproteolysis suggests that the cleavage event may act as a timing switch in the regulation of early versus late T3SS substrates. We also show that YscUC binds to the inner rod protein YscI with a dissociation constant (Kd ) of 3.8 µm and with 1:1 stoichiometry. The significant similarity among different members of the YscU, YscP, and YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria.

Negatively charged lipid membranes promote a disorder-order transition in the Yersinia YscU protein.

The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscUC) domains. Although disordered in solution, inspection of the primary sequence of the linker reveals that positively charged residues are separated with a typical helical periodicity. Here, we demonstrate that the linker sequence of YscU undergoes a largely electrostatically driven coil-to-helix transition upon binding to negatively charged membrane interfaces. Using membrane-mimicking sodium dodecyl sulfate micelles, an NMR derived structural model reveals the induction of three helical segments in the linker. The overall linker placement in sodium dodecyl sulfate micelles was identified by NMR experiments including paramagnetic relaxation enhancements. Partitioning of individual residues agrees with their hydrophobicity and supports an interfacial positioning of the helices. Replacement of positively charged linker residues with alanine resulted in YscUC variants displaying attenuated membrane-binding affinities, suggesting that the membrane interaction depends on positive charges within the linker. In vivo experiments with bacteria expressing these YscU replacements resulted in phenotypes displaying significantly reduced effector protein secretion levels. Taken together, our data identify a previously unknown membrane-interacting surface of YscUC that, when perturbed by mutations, disrupts the function of the pathogenic machinery in Yersinia.

Autoproteolysis and intramolecular dissociation of Yersinia YscU precedes secretion of its C-terminal polypeptide YscU(CC).

Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscU(CC). Here we show that depletion of calcium induces intramolecular dissociation of YscU(CC) from YscU followed by secretion of the YscU(CC) polypeptide. Thus, YscU(CC) behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscU(CC)in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscU(CC) dissociation for Yop secretion. We propose that YscU(CC) orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.

Phosphorylation of proteins involved in activity-dependent forms of synaptic plasticity is altered in hippocampal slices maintained in vitro.

The acute hippocampal slice preparation has been widely used to study the cellular mechanisms underlying activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Although protein phosphorylation has a key role in LTP and LTD, little is known about how protein phosphorylation might be altered in hippocampal slices maintained in vitro. To begin to address this issue, we examined the effects of slicing and in vitro maintenance on phosphorylation of six proteins involved in LTP and/or LTD. We found that AMPA receptor (AMPAR) glutamate receptor 1 (GluR1) subunits are persistently dephosphorylated in slices maintained in vitro for up to 8 h. alpha calcium/calmodulin-dependent kinase II (alphaCamKII) was also strongly dephosphorylated during the first 3 h in vitro but thereafter recovered to near control levels. In contrast, phosphorylation of the extracellular signal-regulated kinase ERK2, the ERK kinase MEK, proline-rich tyrosine kinase 2 (Pyk2), and Src family kinases was significantly, but transiently, increased. Electrophysiological experiments revealed that the induction of LTD by low-frequency synaptic stimulation was sensitive to time in vitro. These findings indicate that phosphorylation of proteins involved in N-methyl-D-aspartate (NMDA) receptor-dependent forms of synaptic plasticity is altered in hippocampal slices and suggest that some of these changes can significantly influence the induction of LTD.