Live cell imaging of the cancer‐related transcription factor RUNX2 during mitotic progression

Abstract

The nuclear matrix bound transcription factor RUNX2 is a lineage-specific developmental regulator that is linked to cancer. We have previously shown that RUNX2 controls transcription of both RNA polymerase II genes and RNA polymerase I dependent ribosomal RNA genes. RUNX2 is epigenetically retained through mitosis on both classes of target genes in condensed chromosomes. We have used fluorescence recovery after photobleaching (FRAP) to measure the relative binding kinetics of EGFP-RUNX2 at transcription sites in the nucleus and nucleoli during interphase, as well as on mitotic chromosomes. RUNX2 becomes more strongly bound as cells go from interphase through prophase, with a doubling of the most tightly bound "immobile fraction". RUNX2 exchange then becomes much more facile during metaphase to telophase. During interphase the less tightly bound pool of RUNX2 exchanges more slowly at nucleoli than at subnuclear foci, and the non-exchanging immobile fraction is greater in nucleoli. These results are consistent with a model in which the molecular mechanism of RUNX2 binding is different at protein-coding and ribosomal RNA genes. The binding interactions of RUNX2 change as cells go through mitosis, with binding affinity increasing as chromosomes condense and then decreasing through subsequent mitotic phases. The increased residence of RUNX2 at mitotic chromosomes may reflect its epigenetic function in "bookmarking" of target genes in cancer cells.

Mitotic Bookmarking: Maintaining Post-Mitotic Reprogramming of Transcription Reactivation

“…The family of RUNX proteins (RUNX1, RUNX2 and RUNX3) is composed of lineage-specific transcription factors essential for hematopoietic, neuronal, gastrointestinal, and osteogenic cell fates. Increased evidence showing that RUNX2 is retained at mitotic chromosomes reflects its epigenetic function in bookmarking of target genes in different cancer cells [26]. The FoxA1 protein was originally identified as a pioneer transcription factor by initial binding to DNA elements for transcription induction [27].…”

The abbreviated pluripotent cell cycle

“…A synergistic relationship between nuclear organization and epigenetic control is reflected by localization of factors that support DNA methylation and histone modifications at promoter regulatory domains. Here, conformational modifications support and influence transcriptional activation and/or suppression by mitotic retention of regulatory complexes at target gene loci during mitosis (e.g., bookmarking) to sustain competency for gene expression in progeny cells (Zaidi et al, 2010; Pockwinse et al, 2011).…”

A role for mitotic bookmarking of SOX2 in pluripotency and differentiation

“…However, some transcription factors do not abide by this rule and remain bound to specific genes on mitotic chromosomes. These so-called mitotic bookmarking transcription factors are involved in physiological processes such as phenotypic maintenance (Zaidi et al 2010(Zaidi et al , 2014Kadauke and Blobel 2013;Festuccia et al 2016) and ribosome biogenesis (Chen et al 2002;Grob et al 2014;Lopez-Camacho et al 2014) as well as pathological events such as oncogenic transformation (Blobel et al 2009;Pockwinse et al 2011;Zaidi et al 2014). However, the contribution of mitotic chromosome retention to these functions remains unknown.…”