Dynamic Control of Multiple Genes Using Chromatin Regulators

In mammalian cells the state of chromatin is tightly liked to gene expression. It is essential to understand the rules that connect them if we want to use chromatin regulators to engineer the epigenome. For instance, how fast can chromatin regulators affect gene expression, how long do their effects last as epigenetic memory, and how far along the chromatin fiber do these effects spread? In fact, these questions go back to some of the first epigenetic phenomena reported in the 1930s, where chromosomal rearrangements in fruit flies resulted in stochastic gene silencing – observed as mottled eyes – resulting from repositioning of genes near repressive chromatin. Due to advances in synthetic biology and single-cell measurements, we can now return to these types of questions with new eyes. In order to quantitatively address these questions, we engineered a set of mammalian cell lines that allow us precisely control the recruitment and release of chromatin regulators at fluorescent reporter genes. We chose a set of chromatin regulators associated with various chromatin modifications: histone methylation (KRAB, EED), histone acetylation (HDAC4), and DNA methylation (DNMT3B). We follow the changes in gene expression in single cells over time using time-lapse microscopy and flow cytometry in multiple murine and human cell types (CHO-K1, mESCs, HEK293T, and K562). We find that recruitment of repressive chromatin regulators leads to stochastic all-or-none silencing. In this manner, chromatin regulators allow us to control the fraction of cells in the population that have a particular gene off by varying the duration of recruitment. Gene reactivation is also stochastic and all-or-none. However, different chromatin regulators are associated with different rates of silencing and reactivation, and thus different levels of epigenetic memory: HDAC4 leads to rapidly reversible silencing, DNMT3B is associated with irreversible memory.