Quantitative gene regulation at the cell population-level can be achieved by two fundamentally different modes of regulation at individual gene copies. A “digital” mode involves binary ON/OFF expression states, with population-level variation arising from the proportion of gene copies in each state, while an “analog” mode involves graded expression levels at each gene copy. At the Arabidopsis floral repressor FLOWERING LOCUS C (FLC), “digital” Polycomb silencing is known to facilitate quantitative epigenetic memory in response to cold. However, whether FLC regulation before cold involves analog or digital modes is unknown. Using quantitative fluorescent imaging of FLC mRNA and protein, together with mathematical modelling, we find that FLC expression before cold is regulated by both analog and digital modes. We observe a temporal separation between the two modes, with analog preceding digital. The analog mode can maintain intermediate expression levels at individual FLC gene copies, before subsequent digital silencing, consistent with the copies switching OFF stochastically and heritably without cold. This switch leads to a slow reduction in FLC expression at the cell population-level. These data present a new paradigm for gradual repression, elucidating how analog transcriptional and digital epigenetic memory pathways can be integrated.

Compartmentalization is a hallmark of cellular systems and an ingredient actively exploited in evolution. It is also being engineered and exploited in synthetic biology, in multiple ways. While these have demonstrated important experimental capabilities, understanding design principles underpinning compartmentalization of genetic circuits has been elusive. We develop a systems framework to elucidate the interplay between the nature of the genetic circuit, the spatial organization of compartments, and their operational state (well-mixed or otherwise). In so doing, we reveal a number of unexpected features associated with compartmentalizing synthetic and template-based circuits. These include (i) the consequences of distributing circuits including trade-offs and how they may be circumvented, (ii) hidden constraints in realizing a distributed circuit, and (iii) appealing new features of compartmentalized circuits. We build on this to examine exemplar applications, which consolidate and extend the design principles we have obtained. Our insights, which emerge from the most basic and general considerations of compartmentalizing genetic circuits, are relevant in a broad range of settings.

How epigenetic memory states are established and maintained is a central question in gene regulation. A major epigenetic process important for developmental biology involves Polycomb-mediated chromatin silencing. Significant progress has recently been made on elucidating Polycomb silencing in plant systems through analysis of Arabidopsis FLOWERING LOCUS C ( FLC ). Quantitative silencing of FLC by prolonged cold exposure was shown to represent an ON to OFF switch in an increasing proportion of cells. Here, we review the underlying all-or-nothing, digital paradigm for Polycomb epigenetic silencing. We then examine other Arabidopsis Polycomb-regulated targets where digital regulation may also be relevant.

Chromatin-mediated transcriptional states are central to gene regulation in development and environmental response, with co-transcriptional processes involved in their establishment. Quantitative regulation of Arabidopsis FLOWERING LOCUS C (FLC) is key to determining reproductive strategy. Low FLC expression underpins rapid-cycling and is established through a transcription-coupled chromatin mechanism. Proximal termination of antisense transcripts is linked to histone 3 lysine 4 demethylation of adjacent chromatin that leads to stable Polycomb Repressive Complex 2 (PRC2) silencing. However, how the termination-induced chromatin environment influences the switch to PRC2 silencing is still unclear. Here, we combine molecular approaches with theory to develop a dynamic mathematical model that incorporates sense/antisense transcription, alternative termination sites, and the interplay of these processes with varying levels of activating (H3K4me1)/silencing (H3K27me3) histone modifications. The model captures different feedback mechanisms between co-transcriptional 3′ processing and chromatin modifications, detailing how proximal co-transcriptional polyadenylation/termination can set the subsequent level of productive transcription via removal of H3K4 monomethylation across the locus. Since transcription universally antagonizes Polycomb silencing, this dictates the degree of antagonism to H3K27me3, thus determining the rate at which Polycomb repression is established. These principles are likely to be central to regulating transcriptional output at many targets and generally relevant for Polycomb silencing in many genomes.