Bmi1 is essential for cerebellar development and is overexpressed in human medulloblastomas.

Overexpression of the polycomb group gene Bmi1 promotes cell proliferation and induces leukaemia through repression of Cdkn2a (also known as ink4a/Arf) tumour suppressors. Conversely, loss of Bmi1 leads to haematological defects and severe progressive neurological abnormalities in which de-repression of the ink4a/Arf locus is critically implicated. Here, we show that Bmi1 is strongly expressed in proliferating cerebellar precursor cells in mice and humans. Using Bmi1-null mice we demonstrate a crucial role for Bmi1 in clonal expansion of granule cell precursors both in vivo and in vitro. Deregulated proliferation of these progenitor cells, by activation of the sonic hedgehog (Shh) pathway, leads to medulloblastoma development. We also demonstrate linked overexpression of BMI1 and patched (PTCH), suggestive of SHH pathway activation, in a substantial fraction of primary human medulloblastomas. Together with the rapid induction of Bmi1 expression on addition of Shh or on overexpression of the Shh target Gli1 in cerebellar granule cell cultures, these findings implicate BMI1 overexpression as an alternative or additive mechanism in the pathogenesis of medulloblastomas, and highlight a role for Bmi1-containing polycomb complexes in proliferation of cerebellar precursor cells.

Bmi1 loss produces an increase in astroglial cells and a decrease in neural stem cell population and proliferation.

The polycomb transcriptional repressor Bmi1 promotes cell cycle progression, controls cell senescence, and is implicated in brain development. Loss of Bmi1 leads to a decreased brain size and causes progressive ataxia and epilepsy. Recently, Bmi1 was shown to control neural stem cell (NSC) renewal. However, the effect of Bmi1 loss on neural cell fate in vivo and the question whether the action of Bmi1 was intrinsic to the NSCs remained to be investigated. Here, we show that Bmi1 is expressed in the germinal zone in vivo and in NSCs as well as in progenitors proliferating in vitro, but not in differentiated cells. Loss of Bmi1 led to a decrease in proliferation in zones known to contain progenitors: the newborn cortex and the newborn and adult subventricular zone. This decrease was accentuated in vitro, where we observed a drastic reduction in NSC proliferation and renewal because of NSC-intrinsic effects of Bmi1 as shown by the means of RNA interference. Bmi1(-/-) mice also presented more astrocytes at birth, and a generalized gliosis at postnatal day 30. At both stages, colocalization of bromodeoxyuridine and GFAP demonstrated that Bmi1 loss did not prevent astrocyte precursor proliferation. Supporting these observations, Bmi1(-/-) neurospheres generate preferentially astrocytes probably attributable to a different responsiveness to environmental factors. Bmi1 is therefore necessary for NSC renewal in a cell-intrinsic mode, whereas the altered cell pattern of the Bmi1(-/-) brain shows that in vivo astrocyte precursors can proliferate in the absence of Bmi1.

Ink4a and Arf differentially affect cell proliferation and neural stem cell self-renewal in Bmi1-deficient mice.

The Polycomb group (PcG) gene Bmi1 promotes cell proliferation and stem cell self-renewal by repressing the Ink4a/Arf locus. We used a genetic approach to investigate whether Ink4a or Arf is more critical for relaying Bmi1 function in lymphoid cells, neural progenitors, and neural stem cells. We show that Arf is a general target of Bmi1, however particularly in neural stem cells, derepression of Ink4a contributes to Bmi1(-/-) phenotypes. Additionally, we demonstrate haploinsufficient effects for the Ink4a/Arf locus downstream of Bmi1 in vivo. This suggests differential, cell type-specific roles for Ink4a versus Arf in PcG-mediated (stem) cell cycle control.

The T-box repressors TBX2 and TBX3 specifically regulate the tumor suppressor gene p14ARF via a variant T-site in the initiator.

The murine tumor suppressor p19(ARF) (p14(ARF) in humans) is thought to fulfill an important protective role in preventing primary cells from oncogenic transformation via its action in the p53 pathway. Several disease-implicated regulators of p19(ARF) are known to date, among which are the T-box genes TBX2, which resides on an amplicon in primary breast tumors, and TBX3, which is mutated in the human developmental disorder Ulnar-Mammary syndrome. Here we identify a variant T-site, matching 13 of 20 nucleotides of a consensus T-site, as the essential TBX2/TBX3-binding element in the human p14(ARF) promoter. Mutant analysis indicates that both the consensus T-box and a C-terminal conserved repression domain are essential for p14(ARF) repression. Whereas the core nucleotides required for interaction of the archetypal T-box protein Brachyury with a consensus T-site are conserved in the variant site, additional flanking nucleotides contribute to the specificity of TBX2 binding. This is illustrated by the inability of TBX1A or Xbra to activate via the variant p14(ARF) T-site. Importantly, this suggests a hitherto unsuspected level of specificity associated with T-box factors and corresponding recognition sites in regulating their target genes in vivo.

TBX-3, the gene mutated in Ulnar-Mammary Syndrome, is a negative regulator of p19ARF and inhibits senescence.

Prolonged culturing of rodent cells in vitro activates p19(ARF) (named p14(ARF) in man), resulting in a p53-dependent proliferation arrest known as senescence. The p19(ARF)-Mdm2-p53 pathway also serves to protect primary cells against oncogenic transformation. We have used a genetic screen in mouse neuronal cells, conditionally immortalized by a temperature-sensitive mutant of SV40 large T antigen, to identify genes that allow bypass of senescence. Using retroviral cDNA expression libraries, we have identified TBX-3 as a potent inhibitor of senescence. TBX-3 is a T-box gene, which is found mutated in the human developmental disorder Ulnar-Mammary Syndrome. We have shown that TBX-3 potently represses expression of both mouse p19(ARF) and human p14(ARF). We have also shown here that point mutants of TBX-3, which are found in Ulnar-Mammary Syndrome, have lost the ability to inhibit senescence and fail to repress mouse p19(ARF) and human p14(ARF) expression. These data suggest that the hypoproliferative features of this genetic disorder may be caused, at least in part, by deregulated expression of p14(ARF).

Bmi-1 regulation of INK4A-ARF is a downstream requirement for transformation of hematopoietic progenitors by E2a-Pbx1.

Loss-of-function alterations of INK4A are commonly observed in lymphoid malignancies, but are consistently absent in pre-B cell leukemias induced by the chimeric oncoprotein E2a-Pbx1 created by t(1;19) chromosomal translocations. We report here that experimental induction of E2a-Pbx1 enhances expression of BMI-1, a lymphoid oncogene whose product functions as a transcriptional repressor of the INK4A-ARF tumor suppressor locus. Bmi-1-deficient hematopoietic progenitors are resistant to transformation by E2a-Pbx1; however, the requirement for Bmi-1 is alleviated in cells deficient for both Bmi-1 and INK4A-ARF. Furthermore, the adverse effects of E2a-Pbx1 on pre-B cell survival and differentiation are partially bypassed by forced expression of p16(Ink4a). These results link E2a-Pbx1 with Bmi-1 on an oncogenic pathway that is likely to play a role in the pathogenesis of human lymphoid leukemias through downregulation of the INK4A-ARF gene.

Stem cells and cancer; the polycomb connection.

Proteins from the Polycomb group (PcG) are epigenetic chromatin modifiers involved in cancer development and also in the maintenance of embryonic and adult stem cells. The therapeutic potential of stem cells and the growing conviction that tumors contain stem cells highlights the importance of understanding the extrinsic and intrinsic circuitry controlling stem cell fate and their connections to cancer.

Cooperative action of Tbx2 and Nkx2.5 inhibits ANF expression in the atrioventricular canal: implications for cardiac chamber formation.

During heart development, chamber myocardium forms locally from the embryonic myocardium of the tubular heart. The atrial natriuretic factor (ANF) gene is specifically expressed in this developing chamber myocardium and is one of the first hallmarks of chamber formation. We investigated the regulatory mechanism underlying this selective expression. Transgenic analysis shows that a small fragment of the ANF gene is responsible for the developmental pattern of endogenous ANF gene expression. Furthermore, this fragment is able to repress cardiac troponin I (cTnI) promoter activity selectively in the embryonic myocardium of the atrioventricular canal (AVC). In vivo inactivation of a T-box factor (TBE)- or NK2-homeobox factor binding element (NKE) within the ANF fragment removed the repression in the AVC without affecting its chamber activity. The T-box family member Tbx2, encoding a transcriptional repressor, is expressed in the embryonic myocardium in a pattern mutually exclusive to ANF, thus suggesting a role in the suppression of ANF. Tbx2 formed a complex with Nkx2.5 on the ANF TBE-NKE, and was able to repress ANF promoter activity. Our data provide a potential mechanism for chamber-restricted gene activity in which the cooperative action of Tbx2 and Nkx2.5 inhibits expression in the AVC.