B-Myb is Critical for Proper DNA Duplication During an Unperturbed S Phase in Mouse Embryonic Stem Cells  

Abstract

A common feature of early embryo cells from the inner cell mass (ICM) and of ESCs is an absolute dependence on an atypical cell cycle in which the G1 phase is shortened to preserve their self-renewing and pluripotent nature. The transcription factor B-Myb has been attributed a role in proliferation, in particular during the G2/M phases of the cell cycle. Intriguingly, B-Myb levels in ICM/ESCs are greater than 100 times compared with those in normal proliferating cells, suggesting a particularly important function for this transcription factor in pluripotent stem cells. B-Myb is essential for embryo development beyond the preimplantation stage, but its role in ICM/ESCs remains unclear. Using a combination of mouse genetics, single DNA fiber analyses and high-resolution three-dimensional (3D) imaging, we demonstrate that B-Myb has no influence on the expression of pluripotency factors, but instead B-Myb ablation leads to stalling of replication forks and superactivation of replication factories that result in disorganization of the replication program and an increase in double-strand breaks. These effects are partly due to aberrant transcriptional regulation of cell cycle proliferation factors, namely c-Myc and FoxM1, which dictate normal S phase progression. We conclude that B-Myb acts crucially during the S phase in ESCs by facilitating proper progression of replication, thereby protecting the cells from genomic damage. Our findings have particular relevance in the light of the potential therapeutic application of ESCs and the need to maintain their genomic integrity. STEM CELLS

B- myb is an essential regulator of hematopoietic stem cell and myeloid progenitor cell development

“…In addition, CMPs and GMPs in which B-myb is disrupted in vitro proliferate more slowly. These results are consistent with previous reports documenting a role for B-myb in S/G 2 /M progression and checkpoint control (20,22,25,46,47). A similar phenotype is also observed in the zebrafish crash&burn (crb) mutant (48), which harbors a loss of function mutation in bmyb.…”

“…S8, only PLK1 and BIRC5 (survivin) were predicted to be differentially expressed. The expression pattern of these genes does not overlap with B-myb KO ESCs, which might suggest a differential use of target genes in LSK + cells, or, simply reflects a greater accumulation of B-mybdeficient LSK + cells in S phase compared with ESCs (22). Given that B-myb-null HSCs display a profound engraftment defect, these studies suggest that loss of B-myb induces changes in gene expression that impair self-renewal and results in aberrant lineage commitment.…”

“…When these gene lists were further analyzed using the ChIP Enrichment Analysis (ChEA) database within ENRICHR (http://amp.pharm.mssm.edu/Enrichr) (26,27), considerable overlap was observed with the promoters of genes that are occupied by transcription factors during the control of maintenance, development, and multipotency of HSCs and progenitor cells as well as embryonic stem cells (ESCs) (Dataset S1) (28)(29)(30)(31)(32)(33)(34). Of these transcription factors, C-MYC and E2F-1 are known to be intricately involved in B-MYB-dependent transcription (22,25,(35)(36)(37)(38), whereas others, such as JARID1A, RUNX1 (AML1), TAL1 (SCL), LMO1, and FLI1, are not as well defined with respect to B-MYB transcriptional activation and repression of its targets (25,39). In addition to the transcription factors listed above, we also detected an overlap with genes that are targets of C-MYB, which is known to regulate promoters that are both distinct from and common with other members of the MYB family (40,41).…”

“…GATA2, on the other hand, was decreased in the absence of B-myb. We also examined the expression pattern of genes that modulate S phase progression and G 2 /M transition, some of which have been implicated in the proliferative defects observed in B-myb-null ESCs (22). Of the genes analyzed in Fig.…”

“…In their steady-state, HSCs are largely quiescent and enter the cell cycle in response to stimuli that trigger differentiation and lineage commitment (18). B-myb has been shown to play a role in the cell cycle progression of multiple cell types and is involved in regulating progression through the S and G 2 /M phases (19)(20)(21)(22)(23)(24)(25). To determine whether loss of B-myb expression led to altered cell cycle kinetics in HSCs, pIpC-treated control and B-myb F/Fcre mice were injected with BrdU and BM was harvested 2 h posttreatment.…”