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. In 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 reveals differential reactivation kinetics for key targets of Brn2 and Ascl1, with transcription onset detected in early (anaphase) versus late (early G1) phases, 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.

Regulated interaction of ID2 with the anaphase-promoting complex links progression through mitosis with reactivation of cell-type-specific transcription

“…Thus, we asked whether the phospho-S5-mediated regulation of ID2 stability through controlled interaction with core APC might provide the cross-talk between the intrinsic mitotic regulatory machinery and reactivation of tissue-specific transcriptional programs by bHLH transcription factors. Interestingly, recent work reported that bHLH transcription factors dissociate from mitotic chromosomes 35 . As the ubiquitously expressed E proteins are obligate partners of all bHLH transcription factors and cannot bind DNA in the presence of ID2 27 , we measured E-protein-mediated transcription with an E-box-luciferase reporter in cells transiting through mitosis.…”

Function of Proneural Genes Ascl1 and Asense in Neurogenesis: How Similar Are They?

“…Live-cell imaging of the germinal layers at the lateral ganglionic eminence (LGE) in the ventral telencephalon described a complex lineage, with Radial Glia (RG) NS cells in the Ventricular Zone (VZ) at the top of a hierarchy that includes apically-dividing short neural precursors (SNPs), sub-apically dividing progenitors (SAPs), and intermediate progenitors (IPs) that divide in the Sub Ventricular Zone (SVZ) (Pilz et al, 2013;García and Harwell, 2017). In the LGE, Ascl1 is excluded from (Gsx2 expressing) RG cells (Roychoudhury et al, 2020), starting to be expressed in apically dividing progenitors, most likely SNPs (Soares et al, 2021) (Figure 2A).…”

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][28][29]. Despite the growing number of TFs reported to display MCB, it remains less clear how this binding is mediated.…”

“…In spite of decreased global histone acetylation levels during mitosis, it is at this stage that the negative electrostatic field of DNA is strongest, as result of high chromatin compaction and H3ser10 phosphorylation throughout chromosomes [ 4 ]. Thus, electrostatic interactions between proteins and DNA play a more prominent role in the context of condensed chromosomes during mitosis, or in DNA-dense and heterochromatic regions in the interphase nucleus [ 8 , 20 ]. Hence, binding of TFs to mitotic chromosomes should be discussed considering the different types of interactions that TFs may engage on mitotic chromatin.…”

“…In a recent study on Brn2 (also termed POU3f2), a point-mutation in a residue predicted to interact with the DNA back-bone (but not DNA bases), abolishes MCB in neural stem cells. Conversely, a phospho-mimetic Brn2 derivative devoid of sequence-specific binding (S362D), retains to large extent its ability to interact with mitotic chromosomes [ 20 ]. On a different scale, a large screening based on live-cell imaging analysis of TFs ectopically expressed in embryonic stem (ES) cells, concluded that approximately 20% out of 500 mouse TFs show MCB ability (albeit to various degrees).…”