Hierarchical reactivation of transcription during mitosis-to-G1 transition by Brn2 and Ascl1 in neural stem cells

Abstract

During mitosis, chromatin condensation is accompanied by a global arrest of transcription. Recent studies suggest transcriptional reactivation upon mitotic exit occurs in temporally coordinated waves, but the underlying regulatory principles have yet to be elucidated. In particular, the contribution of sequence-specific transcription factors (TFs) remains poorly understood. Here we report that Brn2, an important regulator of neural stem cell identity, associates with condensed chromatin throughout cell division, as assessed by live-cell imaging of proliferating neural stem cells. By contrast, the neuronal fate determinant Ascl1 dissociates from mitotic chromosomes. ChIP-seq analysis reveals that Brn2 mitotic-chromosome binding does not result in sequence-specific interactions prior to mitotic exit, relying mostly on electrostatic forces. Nevertheless, surveying active transcription using single-molecule RNA-FISH against immature transcripts, indicates the differential presence of TF near chromatin when exiting mitosis is associated with early (anaphase) versus late (early G1) reactivation of key targets of Brn2 and Ascl1, respectively. Moreover, by using a mitotic-specific dominant negative approach, we show that competing with Brn2 binding during mitotic exit reduces the transcription of its target gene Nestin. Our study shows an important role for differential binding of TFs to mitotic chromosomes, governed by their electrostatic properties, in defining the temporal order of transcriptional reactivation during mitosis-to-G1 transition.

Function and regulation of transcription factors during mitosis-to-G1 transition

“…Live-cell imaging experiments using fluorescently tagged proteins have been a major drive in recent studies characterizing how TFs interact with mitotic chromosomes (box 1). Using this approach, a growing number of TFs has been shown to colocalize with condensed chromosomes during mitosis, a property herein referred to as ‘mitotic chromosome binding’ (MCB) [8,20,27–29]. Despite the growing number of TFs reported to display MCB, it remains less clear how this binding is mediated.…”

“…In some cases, however, MCB is only achieved upon the addition of multiple NLSs, suggesting the need to counteract a much lower electrostatic potential. One example is Ascl1, which displays MCB only when fused with a tandem of six SV40 NLSs [20]. Regardless of the mechanistic basis, to which extent some of these experiments underline a role for the NLS in the context of a native protein is unclear, in particular when tandems of several NLS sequences were used.…”

“…Regardless of the mechanistic basis, to which extent some of these experiments underline a role for the NLS in the context of a native protein is unclear, in particular when tandems of several NLS sequences were used. Moreover, not all NLS sequences promote mitotic chromosome binding in live-cell imaging studies [20,21]. Future studies should better characterize to which extent specific NLS sequences are predictive of mitotic chromosome binding, and whether other types of protein sequences can play a similar role.…”

“…Of note, no differences were observed at TF binding sites with ATAC-seq (investigated in case of ESRR β ), thus not supporting changes of chromatin accessibility as the underlying mechanism of decreased TF retention [49]. Moreover, the MCB of TFs has been correlated with their colocalization at heterochromatic loci enriched for H3K9me3 in interphase [8,20]. Altogether, these observations indicate the importance of addressing how specific histone PTMs (and in particular H3K9me3) may impact the association of TFs with mitotic chromatin, and how this may result from their contribution to the electrostatic field.…”

“…Importantly, TF phosphorylation can at least in some cases dissociate sequence-specific from non-specific binding, helping to explain why not all MCB TFs display bona fide mitotic bookmarking activity. A striking example is that of Brn2, with a phospho-mimetic form devoid of sequence-specific binding (S362D) retaining to large extent its MCB ability in neural stem cells [20]. In the case of Oct4, mitotic-specific phosphorylation by Aurora kinase B inhibits sequence-specific binding to target genes in ES cells, as assessed by ChIP-PCR.…”