Hit and run transcriptional repressors are difficult to catch in the act Manan Shah1, Alister P.W. Funnell1,2, Kate G.R. Quinlan1 and Merlin Crossley1 1School of Biotechnology and Biomolecular Sciences, UNSW Sydney, Sydney, New South Wales, Australia 2Altius Institute for Biomedical Sciences, Seattle, Washington, USA *Corresponding Author: [email protected] Abstract Transcriptional silencing may not necessarily depend on continuous residence of a sequence- specific repressor at a control element and may act via a ‘hit and run’ mechanism. Due to limitations in assays that detect transcription factor (TF) binding, such as ChIP-Seq, this phenomenon may be challenging to detect and therefore its prevalence may be underappreciated. To explore this possibility we analysed erythroid gene promoters that are repressed directly by GATA1 in an inducible system. We found many repressed genes after bound immediately after induction of GATA1 but the residency of GATA1 decreases over time particularly at repressed genes. Furthermore, we show that the repressive mark H3K27me3 is seldom associated with bound repressors, whereas in contrast the active (H3K4me3) histone mark is overwhelmingly associated with TF binding. We hypothesise that during cellular differentiation and development, certain genes are silenced by repressive TFs that subsequently vacate the region. Catching such repressor TFs in the act of silencing via assays such as ChIP- Seq is thus a temporally challenging prospect. The use of inducible systems, epitope tags and alternative techniques may provide opportunities for detecting elusive ‘hit and run’ transcriptional silencing. Keywords hit and run, transcription factor, repressor, ChIP-Seq, transcriptional repression, transcriptional regulation, GATA1 Abbreviations ChIP - chromatin immunoprecipitation; ChIP-Seq - chromatin immunoprecipitation followed by high throughput sequencing; CUT&RUN - Cleavage Under Targets and Release Using Nuclease; FOG - Friend of GATA1; LacI - bacterial lac repressor; PIC – pre-initiation complex; PRC2 - polycomb repressive complex 2; REST/NRSF - RE1-Silencing Transcription Factor/Neuron-Restrictive Silence Factor; TALENs - transcription activator- like effector nucleases; TF - transcription factor; TSS - transcription start site; ZNFs - zinc finger nucleases Introduction Turning on the expression of a gene typically requires the binding of activator transcription factors (TFs) to regulatory DNA sequence elements, such as promoters and distal enhancers. TF binding subsequently leads to the recruitment of activating cofactors, histone modifying enzymes, and ultimately RNA polymerase II and the general TFs, which form the pre-initiation complex (PIC) [1]. Repeated rounds of transcriptional initiation are achieved by sustained occupancy or re-attendance by the activating TFs. Gene silencing, on the other hand, is different in that it is not necessarily dependent on the continuous residence of a sequence-specific repressor at a control element. This is an important distinction, because assays that detect genome-wide TF binding activity, such as ChIP-Seq (Chromatin Immunoprecipitation followed by high throughput sequencing), typically provide a static snapshot of occupancy precisely as it stands at the time-point of assay. For this reason, ChIP-Seq does not necessarily lend itself to the detection of some modes of transcriptional repression. This is an aspect that when overlooked, can skew our understanding of the gene regulatory mechanisms at play in a given cell type. Here we propose that activator TFs, and certain repressor TFs that operate via sustained or frequent binding of a nucleosome-depleted region, are readily amenable to detection by assays such as ChIP-Seq. In contrast, transcriptional repressors that elicit stable, long-term silencing by a ‘hit and run’ mechanism, will be more challenging to detect. This latter category will require dynamic, temporally controlled systems that allow such repressors to be ‘caught in the act’ during their window of silencing. In other words – repressors can act differently to activators. To use an analogy, in some cases, once the gene is locked down, the gaoler can sometimes, but not always, decide to throw away the key. In contrast, where gene activation is concerned, some sort of a key is likely to be evident every time the gene is transcribed. Figure 1. Models of transcriptional repression. a) Sustained TF binding represses the gene and is amenable to detection via ChIP or other TF binding assays. b) A ‘hit and run’ mechanism; The TF is bound initially and may recruit histone modifying enzymes/chromatin remodellers, which package the locus, preventing transcription and further TF binding. Binding is able to be detected initially but not once the locus is tightly packed (heterochromatin). Transcriptional repression and activation may occur through different mechanisms A classic example of a transcriptional repressor is the bacterial lac repressor (LacI) [2–4]. In the absence of lactose, it resides at the promoters of genes required for lactose metabolism and blocks engagement by RNA polymerase. Upon lactose exposure, LacI undergoes allosteric changes that abrogate its ability to bind these promoters, and subsequently, RNA polymerase is recruited and transcription commences. LacI is thus an example of a continuously binding repressor that renders the lac operon poised to dynamically respond to fluctuating metabolic requirements. Similarly, a textbook example of a mammalian transcriptional repressor is REST/NRSF (RE1- Silencing Transcription Factor/Neuron-Restrictive Silence Factor). REST resides at the control elements of neuronal genes in non-neuronal cells and represses their transcription [5–7]. In a very broad sense, REST thus silences gene expression in a similar fashion to LacI; it remains bound to its target genes for the duration of silencing, a characteristic that is amenable to ready detection. As such, REST is often invoked as a paradigmatic example of a mammalian transcriptional repressor. Yet how generally representative is REST of the mechanism of action of mammalian repressors? Do all sequence-specific transcriptional repressors remain bound to the control elements of the genes they silence throughout the life of the cell? Or rather, is it that we are prone to preferentially detect repressors that operate through this mode by assays such as ChIP- Seq? In somatic eukaryotic cells, only a fraction of the transcriptome is selectively expressed [8,9] and this in turn defines cell type identity. For any given cell type, thousands of genes are silent or repressed, a state that may be preserved through rounds of cell division or differentiation. Some of these may be continuously repressed by resident transcription factors akin to REST. It is tempting to speculate that these genes are in effect reversibly repressed, or poised to respond to specific stimuli, and that displacement of the resident TF could lead to their activation. Other genes, however, are stably repressed by general mechanisms of chromatin silencing. For instance, they may be silenced by local Polycomb-mediated H3K27 trimethylation and formation of facultative heterochromatin [10]. Alternatively, nucleosomal occlusion following remodeling may prevent the access of requisite activating TFs that have limited or no pioneering activity. Indeed, an important distinction to make is that in eukaryotes, as opposed to prokaryotes, the baseline state of chromatin is transcriptionally restrictive [11,12]. That is to say, in vivo, eukaryotic core promoters are intrinsically inert and are occluded by nucleosomes. Transcriptional initiation requires the coordinated action of transcriptional activators to displace nucleosomes and recruit the PIC. The ground state is thus one of transcriptional inactivity and the repressive capacity of nucleosomes alone or facultative heterochromatin can maintain gene silencing long after the departure of the repressor(s) that elicited the initial silencing. While sequence-specific repressors may be required to initially silence an active gene, they may not be essential for durable propagation of the repressed state. This mechanism of action is described as ‘hit and run’. These stable repression mechanisms are particularly important during development, enabling perpetuation of gene expression patterns through cell divisions and cellular differentiation. The patterning of Hox gene expression in Drosophila early embryogenesis is well studied and provides useful insights. During early embryogenesis, locally expressed gap gene products (transcriptional activators and repressors) directly bind to cis-acting regulatory regions in Hox genes and regulate transcription. The Hox genes that are initially repressed become stably repressed for the rest of development even when the gap repressors are no longer present. This repression is maintained by factors of the Polycomb group (PcG) [13–15]. Removal of most PcG proteins from proliferating cells causes de-repression. Interestingly, the resupply of these proteins within a certain window of time can cause re-repression [16]. This suggests that a mechanism beyond simple nucleosomal occlusion may be required for true long-term repression across multiple cell divisions or development. The maintainence of repression even after the initial transcriptional repressors are no longer present is consistent with a ‘hit and run’ mechanism. Where might one expect to detect evidence for ‘hit and run’ repressors? Many TFs appear to