ABSTRACT
Master transcription factors have the ability to direct and reverse cellular identities, and consequently their genes must be subject to particular transcriptional control. However, it is unclear which molecular processes are responsible for impeding their activation and safeguarding cellular identities. Here we show that the targeting of dCas9-VP64 to the promoter of the master transcription factor Sox1 results in strong transcript and protein up-regulation in neural progenitor cells (NPCs). This gene activation restores lost neuronal differentiation potential, which substantiates the role of Sox1 as a master transcription factor. However, despite efficient transactivator binding, major proportions of progenitor cells are unresponsive to the transactivating stimulus. By combining the transactivation domain with epigenome editing we find that among a series of euchromatic processes, the removal of DNA methylation (by dCas9-Tet1) has the highest potential to increase the proportion of cells activating foreign master transcription factors and thus breaking down cell identity barriers.
INTRODUCTION
Cellular features, including identity, behavior, and potency, are initiated during development by transcriptional programs, which are triggered by individual master transcription factors1. These proteins are temporally and spatially highly restricted in their expression. The erroneous expression of developmental master transcription factors is a permanent threat to the integrity of any multicellular organism, as they are able to impose their transcriptional programs outside of their natural context. This issue has been exemplified by the so-called reprogramming factors, which are master transcription factors able to overwrite already established cellular programs by enforcing foreign cell identities2. Because cell identity switches rarely (if at all) occur under normal circumstances, it is likely that special measures restrict the activation of master transcription factors in somatic cells.
Although it is obvious that the most effective barriers against cell identity changes would control the expression of master transcription factor genes and that several chromatin processes (e.g., DNA methylation, polycomb, nuclear topology) have been implicated in this process, causalities were extremely difficult to establish. This is due to the fact that until recently the lack of experimental options for site-specific manipulation made it challenging to generate unambiguous experimental evidence for the causality of local chromatin features. But even with recent advancements in the field of DNA targeting, most experimental reprogramming approaches still rely on vector-mediated overexpression of fate determinants, bypassing the primary activation of the endogenous reprogramming factor gene. Thus, the potentially most relevant cell identity barriers have evaded identification and functional analysis.
However, two experimental strategies, both based on adaptations of the bacterial CRISPR complex, have recently enabled the circumvention of these two issues and can be used to investigate the presence and identity of such functional barriers: transcriptional engineering, which is the manipulation of gene expression by targeting artificial transcription factors to gene promoters3,4,5, as well as epigenome editing, which is the site-specific manipulation of epigenomic features, for which a number of options have been developed recently6,7. Gene induction by transcriptional engineering aims for the targeted manipulation of gene expression of endogenous genes8. While the epigenomic and/or transcriptional effects are defined by the specific dCas9 fusion protein used, the target locus is specified in the protospacer sequence encoded in the short synthetic guide RNAs (gRNAs). The system most frequently used to date to achieve gene activation is a fusion of an enzymatically dead version of Cas9 with four copies of the transactivator domain of the viral transcription factor VP16 (dCas9-VP64)9. Transcriptional engineering has been successfully used for transcription activation of a number of genes, including master transcription factors like MyoD, Ascl1, and Sox25,10,11. Although examples remain sparse and we lack comprehensive approaches, it emerges already that endogenous master transcription factors behave in a particularly rigid manner compared to control genes5,12,13.
During neurogenesis, a temporal sequence of master transcription factors controls the progression of neural development. The sex-determining region Y-box transcription factor 1 (Sox1) is amongst the first transcription factors induced during neural development in xenopus14, chicken15, rodents16, and humans17. In mice, Sox1 shows strong and specific expression in neuro-epithelial cells18,19, the progenitors of all neural cells. Sox1 has some sequence identity (51%) to Sox2, but unlike its paralog it is not expressed in pluripotent cells and only becomes detectable in the newly formed neuroectoderm around the onset of somitogenesis20. Shortly thereafter, Sox1 expression disappears, except in the adult neural stem cell niches of the hippocampus and the adult subventricular zone, where it marks a population of progenitor cells with long-term neurogenic potential21. Interestingly, Sox1-positive NSCs can be propagated only poorly in vitro, as cultured cells irreversibly lose Sox1 expression22. This conversion coincides with a progressive loss of neuronal differentiation potential and parallels the natural development in vivo. Despite its undeniable relevance as an early lineage marker, the functional roles of Sox1 are, compared to its paralogs Sox2, Sox9, Sox10 and Sry, still poorly understood23.
Here we show that Sox1 is a master cell identity factor: the induction of its endogenous gene copy restores the neuronal differentiation potential of glial progenitor cells. Moreover, chromatin features, in particular DNA methylation at its promoter, tightly control the capability of Sox1 to be trans-activated. By combining epigenome editing and transcriptional engineering, we demonstrate that the selective removal of this barrier increases the number of responsive cells significantly, proving the causal role of the chromatin mark.
