A dual-purpose real-time indicator and transcriptional integrator for calcium detection in living cells

Calcium is a ubiquitous second messenger in eukaryotes, correlated with neuronal activity and T-cell activation among other processes. Real-time calcium indicators such as GCaMP have recently been complemented by newer calcium integrators that convert transient calcium activity into stable gene expression. Here we introduce LuCID, a dual-purpose real-time calcium indicator and transcriptional calcium integrator which combines the benefits of both calcium detection technologies. We show that the calcium-dependent split luciferase component of LuCID provides real-time bioluminescence readout of calcium dynamics in cells, while the GI/FKF1 split GAL4 component of LuCID converts bioluminescence into stable gene expression. We also show that LuCID modular design enables it to also be used for dual-purpose detection of other cellular events such as protein-protein interactions. LuCID should facilitate the study of cell populations and circuits that utilize calcium for signaling.

Lactylation, a Novel Metabolic Reprogramming Code: Current Status and Prospects

Lactate is an end product of glycolysis. As a critical energy source for mitochondrial respiration, lactate also acts as a precursor of gluconeogenesis and a signaling molecule. We briefly summarize emerging concepts regarding lactate metabolism, such as the lactate shuttle, lactate homeostasis, and lactate-microenvironment interaction. Accumulating evidence indicates that lactate-mediated reprogramming of immune cells and enhancement of cellular plasticity contribute to establishing disease-specific immunity status. However, the mechanisms by which changes in lactate states influence the establishment of diverse functional adaptive states are largely uncharacterized. Posttranslational histone modifications create a code that functions as a key sensor of metabolism and are responsible for transducing metabolic changes into stable gene expression patterns. In this review, we describe the recent advances in a novel lactate-induced histone modification, histone lysine lactylation. These observations support the idea that epigenetic reprogramming-linked lactate input is related to disease state outputs, such as cancer progression and drug resistance.

Lineage Tracing in Chronic Lymphocytic Leukemia Reveals Clones with Stable Gene Expression States That Differentially Respond to Therapy

Cancer's ability to evolve and adapt is a major challenge to therapeutic success. Fueling this evolution is vast tumor heterogeneity, with constituent clones varying in their genetics, epigenetics and response to therapy. As a field, however, we have yet to couple the genetic alterations present within individual clones to their transcriptional or functional outputs. Here, we applied a novel adaptation of clone tracing that integrates DNA barcoding with single-cell RNA sequencing (scRNA-seq) to HG3, a CLL cell line harboring del(13q) and no other known cancer drivers, to model in vitro responses to front-line chemotherapy with fludarabine and cyclophosphamide at clone-level resolution. To generate a high-diversity barcode library compatible with scRNA-seq, a random pool of 20 base pair DNA barcodes was introduced into the 3'UTR of a reporter gene in a lentiviral expression vector. This viral barcode library was transduced into HG3 cells (at MOI 0.1 to minimize multiple barcode-tagging of cells) and 1.2x106 barcoded cells were sorted and expanded to establish the parental barcoded HG3 population. We subsequently treated this barcoded population with an LD95 combined dose of fludarabine and mafosfamide (the in vitro analog of cyclophosphamide) in 8 parallel experiments. Cell barcodes were sequenced prior to treatment (TP1) and following outgrowth from treatment (TP2) for analysis of clonal composition. 10,000 cells each from TP1 and from 2 of 8 parallel replicates at TP2 were processed for scRNA-seq. We observed a massive decrease in viability across all 8 replicates, with regrowth occurring at 20 days post-treatment. Barcode analysis revealed a marked decrease in clonal diversity from TP1 to TP2 (11,827 to 2,622 ± 380, n=8; or ~78%), and clones that survived treatment did so consistently such that 94% of surviving cells in each replicate had a clonal identity that was present in all 8 replicates. Analysis of clonally-resolved transcriptional profiles revealed that clones consistently fell into one of two stable gene expression states (clusters) prior to treatment, with nominal intermixing between populations. Treatment predominantly selected for clones comprising the smaller of these two clusters (TP1-'high tolerance'), with only a minimal number of resistant clones originating from the larger cluster at TP1 (TP1-'low tolerance'). Pathway and gene set enrichment analysis of these two TP1 clusters demonstrated that TP1-high tolerance had a stark upregulation of common CLL signaling pathways (i.e. WNT and CXCR4, an inflammatory/migratory phenotype) and a reliance on chromatin modification pathways. TP1-low tolerance, on the other hand, exhibited upregulated type 2 antigen presentation and prostaglandin biosynthesis/metabolism which has a known role in driving inflammation and migration in adjacent cells (Wang et al, BMJ 2006). These gene expression states remained stable after treatment, but with the added upregulation of well-described mechanisms of resistance to cyclophosphamide, with TP1-high tolerance exhibiting upregulation of GSTP1, a glutathione S-transferase that is a main mediator of cyclophosphamide metabolism, and TP1-low tolerance exhibiting upregulation of members of the ALDH family thought to ameliorate toxicity from chemotherapy-induced ROS (Andersson et al, Acta Oncologica 1995). Through this work, we resolved the underlying clonal composition of a CLL cell line and observed that the constituent clones exhibit stable and discrete gene expression states that differentially respond to chemotherapy. We noted two different axes of resistance - a more successful avenue that relies on WNT and CXCR4 signaling as well as cyclophosphamide clearance for resistance, and a less-successful avenue that involves clearance of reactive oxygen species for survival. The intersection of these two critical CLL pathways in in vitro resistance to first-line CLL therapy is of particular interest given the FAT1 (WNT regulator) mutations and CXCR4 upregulation frequently seen in chemo-refractory CLL (Messina et al, Blood 2014; Burger et al, Blood 2006), and future efforts will assess the stability and interplay of these two pathways in patient samples collected upon relapse to fludarabine and cyclophosphamide. Further, our approach provides a template for the high-resolution study of tens of thousands of clones and their respective phenotypes in a mixed leukemic population. Disclosures Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Pharmacyclics: Research Funding; Neon Therapeutics: Other: Member, Advisory Board.

WUSCHEL acts as an auxin response rheostat to maintain apical stem cells in Arabidopsis

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.

Reconstructing an epigenetic landscape using a genetic ‘pulling’ approach

Cells use genetic switches to shift between alternate stable gene expression states, e.g., to adapt to new environments or to follow a developmental pathway. Conceptually, these stable phenotypes can be considered as attractive states on an epigenetic landscape with phenotypic changes being transitions between states. Measuring these transitions is challenging because they are both very rare in the absence of appropriate signals and very fast. As such, it has proven difficult to experimentally map the epigenetic landscapes that are widely believed to underly developmental networks. Here, we introduce a new nonequilibrium perturbation method to help reconstruct a regulatory network’s epigenetic landscape. We derive the mathematical theory needed and then use the method on simulated data to reconstruct the landscapes. Our results show that with a relatively small number of perturbation experiments it is possible to recover an accurate representation of the true epigenetic landscape. We propose that our theory provides a general method by which epigenetic landscapes can be studied. Finally, our theory suggests that the total perturbation impulse required to induce a switch between metastable states is a fundamental quantity in developmental dynamics.

Seasonal Stability and Dynamics of DNA Methylation in Plants in a Natural Environment

: DNA methylation has been considered a stable epigenetic mark but may respond to fluctuating environments. However, it is unclear how they behave in natural environments. Here, we analyzed seasonal patterns of genome-wide DNA methylation in a single clone from a natural population of the perennial Arabidopsis halleri. The genome-wide pattern of DNA methylation was primarily stable, and most of the repetitive regions were methylated across the year. Although the proportion was small, we detected seasonally methylated cytosines (SeMCs) in the genome. SeMCs in the CHH context were detected predominantly at repetitive sequences in intergenic regions. In contrast, gene-body CG methylation (gbM) itself was generally stable across seasons, but the levels of gbM were positively associated with seasonal stability of RNA expression of the genes. These results suggest the existence of two distinct aspects of DNA methylation in natural environments: sources of epigenetic variation and epigenetic marks for stable gene expression.

WUSCHEL acts as a rheostat on the auxin pathway to maintain apical stem cells in Arabidopsis

ABSTRACTTo maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.