Gene-profiling studies suggest that
Bmi1 modulates HSC self-renewal through the regulation of genes important for stem cell fate decisions, as well as survival genes, antiproliferative genes, and stem cell–associated genes (Figure ) (
25). The previously mentioned
Bmi1 target, the
Ink4a locus (
28), encodes
p16Ink4a and
p19Arf using different promoters (
38). Enforced expression of
p16Ink4a and
p19Arf in HSCs led to senescence and apoptosis, respectively (
25). In neural stem cells,
p16Ink4a deficiency partially restored the ability of
Bmi1-deficient stem cells to self-renew (
26). Figure illustrates regulation of the cell cycle and senescence by p16
Ink4a and p19
Arf. During the cell cycle, pRB is hyperphosphorylated by the cyclin D/cyclin-dependent kinases 4 and 6 (cyclin D/Cdk4/6) complex (
39). The hyperphosphorylated pRB is unable to bind and inhibit E2F transcription factor, allowing transcription of E2F target genes that are important for the G1/S transition, such as DNA polymerase II, cyclin E, p19, myb, and dihydrofolate reductase (
40). This allows cell cycle progression. In the absence of Bmi1, p16
Ink4a is upregulated and prevents binding of Cdk4/6 to cyclin D, inhibiting the kinase activity. This results in hypophosphorylated pRB, which then binds E2F and inhibits E2F-mediated transcription, leading to cell cycle arrest and senescence (
39). p19
Arf sequesters mouse double minute 2 (MDM2) and inhibits p53 degradation, resulting in p53-mediated cell cycle arrest and apoptosis (
41,
42). Point mutations and deletion of
p16Ink4a and
p19Arf are frequently found in many types of human cancers, which implicates them as key regulators of immortalization and/or senescence checkpoints.
Mice lacking
Bmi1 showed induction of both
p16Ink4a and
p19Arf in various hematopoietic and neuronal tissues (
25). Overexpression of
p16Ink4a and
p19Arf in adult HSCs induced cell cycle arrest and apoptosis via the pRB and the p53-dependent pathway, respectively. Double deletion of the
Bmi1 and
p16Ink4a/p19Arf genes partially rescued the phenotypes observed in
Bmi1-deficient mice (
28), suggesting that
p16Ink4a,
p19Arf, and p53 are downstream effectors of
Bmi1 that are involved in the control of the proliferation and survival of HSCs during self-renewing cell divisions (Figure ). Therefore,
Bmi1 maintains the HSC pool in part by repressing genes involved in cellular senescence. Increased expression of the p53 target gene
Wig1 in
Bmi1–/– bone marrow suggests that the
p19Arf pathway may have been activated in
Bmi1–/– hematopoietic cells. Wig1 is a double-stranded RNA-binding protein and inhibits tumor growth in vitro, suggesting that it may function in stress-induced p53 responses (
43). The observation that p53-deficient mice have increased numbers of stem cells is consistent with the notion that p53 might be a downstream effector of Bmi1 (
44). In addition, some of the
Hox9 family of genes are also affected in
Bmi1-deficient hematopoietic tissues and neurospheres (
25,
26). Determination of the relative contribution of each of these pathways to the regulation of HSC self-renewal will require careful analysis of the HSCs from double- or triple-knockout mice.
There is evidence that
Bmi1 might regulate telomerase expression in human mammary epithelial cells (MECs) and might play a role in the development of human breast cancer.
Bmi1 is overexpressed in several breast cancer cell lines and postselection human MECs immortalized with human papilloma virus
E6 oncogene, which abrogates the p53/p21
waf pathway (
45), suggesting that
Bmi1 might be involved in immortalization. Postselection MECs can be obtained by regular feeding of a heterogeneous population of MECs from primary mammary tissue. During this process, the
p16Ink4a gene is progressively silenced and not expressed in postselection MECs (
46,
47). Overexpression of
Bmi1 in postselection MECs bypasses senescence, extending replicative lifespan and immortalizing MECs. This is associated with human telomerase reverse transcriptase (hTERT) expression, which leads to induction of telomerase activity. Although hTERT is a direct target of c-Myc–induced transcription in MECs (
48,
49),
Bmi1 appeared to act independently of c-Myc. Since
Bmi1 is a transcription repressor, induction of telomerase is probably mediated by an indirect mechanism. Deletion analysis of the Bmi1 protein suggested that the
RING finger, as well as the conserved helix-turn-helix domain, was required for its ability to induce telomerase and immortalization. These data suggest that
Bmi1 directly or indirectly regulates telomerase expression in MECs and might play a role in the development of human breast cancer. However,
Bmi1 induction of telomerase is cell type specific;
Bmi1 fails to induce telomerase in fibroblasts (
45). This is consistent with the observation that
Bmi1 overexpression did not immortalize human fibroblasts (
37). It is not known whether
Bmi1 is involved in telomere function in normal breast stem cells.
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