Negative Autoregulation Controls size Scaling in Confined Gene Expression Reactions

Gene expression via transcription-translation is the most fundamental reaction to sustain biological systems, and complex reactions such as this one occur in a small compartment of living cells. Transcriptional feedback that controls gene expression during mRNA synthesis is a vital mechanism that regulates protein synthesis in cells. There is increasing evidence that the cellular compartment induces steric effects in gene expression reactions. However, the finite-size effect of spatial constraints on feedback regulation is not well understood. Here, we study the confinement effect on transcriptional negative feedback regulation of gene expression reactions using a theoretical model. We find that negative feedback regulation alters the scaling relation of gene expression level on compartment volume, approaching the regular scaling relation without the steric effect. Our findings suggest that negative autoregulatory feedback at the transcription step can dampen the size-dependence of protein expression levels in heterogeneous cell populations.

Using computational modelling to reveal mechanisms of epigenetic Polycomb control

The Polycomb system is essential for stable gene silencing in many organisms. This regulation is achieved in part through addition of the histone modifications H3K27me2/me3 by Polycomb Repressive Complex 2 (PRC2). These modifications are believed to be the causative epigenetic memory elements of PRC2-mediated silencing. As these marks are stored locally in the chromatin, PRC2-based memory is a cis-acting system. A key feature of stable epigenetic memory in cis is PRC2-mediated, self-reinforcing feedback from K27-methylated histones onto nearby histones in a read-write paradigm. However, it was not clear under what conditions such feedback can lead to stable memory, able, for example, to survive the perturbation of histone dilution at DNA replication. In this context, computational modelling has allowed a rigorous exploration of possible underlying memory mechanisms and has also greatly accelerated our understanding of switching between active and silenced states. Specifically, modelling has predicted that switching and memory at Polycomb loci is digital, with a locus being either active or inactive, rather than possessing intermediate, smoothly varying levels of activation. Here, we review recent advances in models of Polycomb control, focusing on models of epigenetic switching through nucleation and spreading of H3K27me2/me3. We also examine models that incorporate transcriptional feedback antagonism and those including bivalent chromatin states. With more quantitative experimental data on histone modification kinetics, as well as single-cell resolution data on transcription and protein levels for PRC2 targets, we anticipate an expanded need for modelling to help dissect increasingly interconnected and complex memory mechanisms.

Proteomic analysis of Drosophila CLOCK complexes identifies rhythmic interactions with SAGA and Tip60 complex component NIPPED-A

Circadian clocks keep time via ~ 24 h transcriptional feedback loops. In Drosophila, CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors are feedback loop components whose transcriptional status varies over a circadian cycle. Although changes in the state of activators and repressors has been characterized, how their status is translated to transcriptional activity is not understood. We used mass spectrometry to identify proteins that interact with GFP-tagged CLK (GFP-CLK) in fly heads at different times of day. Many expected and novel interacting proteins were detected, of which several interacted rhythmically and were potential regulators of protein levels, activity or transcriptional output. Genes encoding these proteins were tested to determine if they altered circadian behavior via RNAi knockdown in clock cells. The NIPPED-A protein, a scaffold for the SAGA and Tip60 histone modifying complexes, interacts with GFP-CLK as transcription is activated, and reducing Nipped-A expression lengthens circadian period. RNAi analysis of other SAGA complex components shows that the SAGA histone deubiquitination (DUB) module lengthened period similarly to Nipped-A RNAi knockdown and weakened rhythmicity, whereas reducing Tip60 HAT expression drastically weakened rhythmicity. These results suggest that CLK-CYC binds NIPPED-A early in the day to promote transcription through SAGA DUB and Tip60 HAT activity.

Lamin A/C Deficiency Enables Increased Myosin2 Bipolar Filament Ensembles Which Promote Divergent Actomyosin Network Anomalies Through Self Organization

Nuclear envelope proteins influence cell cytoarchitecure by poorly understood mechanisms. Here we show that siRNA-mediated silencing of lamin A/C (LMNA) promotes contrasting stress fiber assembly and disassembly in individual cells and within cell populations. We show that LMNA deficient cells have elevated myosin-II bipolar filament accumulations, irregular formation of actin comet tails and podosome-like adhesions, increased steady state nuclear localization of the mechanosensitive transcription factors MKL1 and YAP, and induced expression of some MKL1/Serum Response Factor (SRF) regulated genes such as that encoding myosin-IIA (MYH9). Our studies utilizing live cell imaging and pharmacological inhibition of myosin-II, support a mechanism of deregulated myosin-II self-organizing activity at the nexus of divergent actin cytoskeletal aberrations resultant from LMNA loss. In light of our results, we propose a model of how the nucleus, via linkage to the cytoplasmic actomyosin network, may act to control myosin-II contractile behavior through both mechanical and transcriptional feedback mechanisms.