Lineage-Specific Mitotic Bookmarking by Hematopoietic Transcription Factor GATA1

Abstract 547

Hematopoietic lineage choice decisions are stably maintained throughout many cell divisions. For example, erythroid precursor cells undergo several rounds of cell division during their maturation. During each mitosis, most transcription factors separate from chromatin causing transcription to cease globally. Mitosis therefore poses a challenge for transcription factors to re-associate with the appropriate target sites in chromatin of newborn cells. The epigenetic mechanisms that cement lineage stability and resist cell reprogramming during mitosis are poorly understood, although recent evidence supports the existence of “bookmarking” factors that remain bound to mitotic chromatin.

Since the hematopoietic transcription factor GATA1 controls the expression of essentially all erythroid-specific genes, we asked whether it might play a role in maintaining erythroid gene expression programs throughout the cell cycle. Live cell confocal imaging revealed that foci of high GATA1 density are present within mitotic chromatin. Using a novel approach that combines mitotic cell sorting with ChIP-Seq, we defined mitotic GATA1 binding sites on a genome-wide scale. Remarkably, whereas GATA1 vacated the great majority of its target sites during mitosis, including the archetypical GATA1 regulated genes α- and β-globin, those target sites where GATA1 was maintained during mitosis showed a strong tendency to reside near genes encoding key developmental regulators of hematopoiesis (e.g., Zfpm1, Nfe2, Klf1, Gata1, Gata2, Runx1). Tissue-specific GATA1 co-regulators such as FOG-1 and the SCL complex dissociated from GATA1-occupied elements during mitosis, suggesting that GATA1 persists at these sites to facilitate their spatially and temporally appropriate reassembly upon exit from mitosis. Consistent with the notion that GATA1 acts as a mitotic bookmark for its mitotic target genes, timed primary transcript analysis revealed that genes that are marked by GATA1 during mitosis re-activate more rapidly upon G1 entry than those that are not.

To directly address the functional importance of mitotic chromatin binding, we developed a version of GATA1 that is selectively degraded during mitosis but remains stable during interphase. This strategy allowed us to prove, for the first time, that the presence of a transcription factor is required specifically during mitosis for timely reactivation of its mitotic target genes. In addition, mitotically disrupted GATA1 failed to fully repress markers of immature erythroid precursors (e.g., Kit, Lyl1), highlighting a potential role of mitotic GATA1 bookmarking for establishing and maintaining lineage- and developmental stage-specific transcriptional programs.

Follow-up mechanistic experiments to define the mode by which GATA1 operates during mitosis are underway and will be discussed at the meeting. Together, these studies establish GATA1 as a bona fide mitotic bookmarking factor and provide a deeper understanding by which transcription programs are faithfully perpetuated through cell divisions to maintain lineage stability.