Repression of the Antioxidant Pyrroloquinoline Quinone in Skin Aging Induced by Bmi-1 Deficiency.

It is uncertain whether Bmi-1 deficiency could lead to skin aging by redox imbalance and DNA damage. In this study, we first confirmed that Bmi-1 had a relatively high expression level in the skin and Bmi-1 expression levels gradually decreased with age. Then, we studied the role of Bmi-1 in the skin using a Bmi-1-/- mouse model. Bmi-1-/- mice were supplemented with or without pyrroloquinoline quinone (PQQ) for 5 weeks, and their skin phenotypes were compared with Bmi1-/- and wild-type littermates. Our results showed that Bmi-1-/- mice displayed decreased vertical thickness of skin, sparse hair follicles, and thinner and more irregular collagen bundles. Mechanistically, increased oxidative stress with reducing antioxidant capacity and induced DNA damage occurred in Bmi-1-/- mice. Subsequently, this would lead to reduced cell proliferation, increased cell senescence and matrix metalloproteinases (MMPs), and the degradation of fibroblast function and further reduce collagen synthesis. All pathological alterations in the skin of Bmi-1-/- mice were alleviated by PQQ supplementation. These results demonstrated that Bmi-1 might play a key role in protection from skin aging by maintaining redox balance and inhibiting DNA damage response and will be a novel and potential target for preventing skin aging.

PHC1 maintains pluripotency by organizing genome-wide chromatin interactions of the Nanog locus.

Polycomb group (PcG) proteins maintain cell identity by repressing gene expression during development. Surprisingly, emerging studies have recently reported that a number of PcG proteins directly activate gene expression during cell fate determination process. However, the mechanisms by which they direct gene activation in pluripotency remain poorly understood. Here, we show that Phc1, a subunit of canonical polycomb repressive complex 1 (cPRC1), can exert its function in pluripotency maintenance via a PRC1-independent activation of Nanog. Ablation of Phc1 reduces the expression of Nanog and overexpression of Nanog partially rescues impaired pluripotency caused by Phc1 depletion. We find that Phc1 interacts with Nanog and activates Nanog transcription by stabilizing the genome-wide chromatin interactions of the Nanog locus. This adds to the already known canonical function of PRC1 in pluripotency maintenance via a PRC1-dependent repression of differentiation genes. Overall, our study reveals a function of Phc1 to activate Nanog transcription through regulating chromatin architecture and proposes a paradigm for PcG proteins to maintain pluripotency.

Bmi-1 determines the stemness of renal stem or progenitor cells.

Renal stem or progenitor cells (RSCs), labeled with CD24 and CD133, play an important role during the repair of renal injury. Bmi-1 is a critical factor in regulating stemness of adult stem cells or progenitor cells. To investigate whether Bmi-1 determines the stemness of RSCs by inhibiting p16 and p53, and/or maintaining redox balance, RSCs were isolated, cultured and analyzed for stemness characterizations. In RSCs from Bmi-1-deficient (Bmi-1-/-) mice and wild type (WT) littermates, self-renewal, stemness, and expressions of molecules for regulating redox balance and cell cycle progression were compared. Self-renewal of RSCs from Bmi-1 and p16 double-knockout (Bmi-1-/-p16-/-), Bmi-1 and p53 double-knockout (Bmi-1-/-p53-/-) and N-acetylcysteine (NAC)-treated Bmi-1-/- mice were further analyzed for amelioration. Human renal proximal tubular epithelial cells (HK2) were also used for signaling analysis. Our results showed that third-passage RSCs from WT mice had good stemness; Bmi-1 deficiency led to the decreased stemness, and the increased apoptosis for RSCs; NAC treatment or p16/p53 deletion ameliorated the decreased self-renewal of RSCs in Bmi-1 deficiency mice by maintaining redox balance or inhibiting cell cycle arrest respectively; Oxidative stress (OS) could negatively feedback regulate the mRNA expressions of Bmi-1, p16 and p53. In conclusion, Bmi-1 determined the stemness of RSCs through maintaining redox balance and preventing cell cycle arrest. Thus, Bmi-1 signaling molecules would be novel therapeutic targets for maintaining RSCs and hampering the progression of kidney diseases to prevent renal failure.

Bmi deficiency causes oxidative stress and intervertebral disc degeneration which can be alleviated by antioxidant treatment.

The transcriptional repressor Bmi-1 is involved in cell-cycle regulation and cell senescence, the deficiency of which has been shown to cause oxidative stress. This study investigated whether Bmi-1 deficiency plays a role in promoting disc degeneration and the effect of treatment with antioxidant N-acetylcysteine (NAC) on intervertebral disc degeneration. Bmi-1-/- mice were treated with the antioxidant NAC, supplied in drinking water (Bmi-1-/- +NAC). For in vitro experiments, mouse intervertebral discs were cultured under low oxygen tension and serum-limiting conditions in the presence of tumour necrosis factor α and interleukin 1ß in order to mimic degenerative insult. Disc metabolism parameters in these in vitro and in vivo studies were evaluated by histopathological, immunohistochemical and molecular methods. Bmi-1-/- mice showed lower collagen Ⅱ and aggrecan levels and higher collagen Ⅹ levels than wild-type and Bmi-1-/- +NAC mice. Bmi-1-/- mice showed significantly lower superoxide dismutase (SOD)-1, SOD-2, glutathione peroxidase (GPX)-1 and GPX-3 levels than their wild-type littermates and Bmi-1-/- + NAC mice. Relative to Bmi-1-/- mice, the control and Bmi-1-/- +NAC mice showed significantly lower p16, p21, and p53 levels. These results demonstrate that Bmi-1 plays an important role in attenuating intervertebral disc degeneration in mice by inhibiting oxidative stress and cell apoptosis.

Bmi1 restricts the adipogenic differentiation of bone marrow stromal cells to maintain the integrity of the hematopoietic stem cell niche.

The polycomb group protein Bmi1 maintains hematopoietic stem cell (HSC) functions. We previously reported that Bmi1-deficient mice exhibited progressive fatty changes in bone marrow (BM). A large portion of HSCs reside in the perivascular niche created partly by endothelial cells and leptin receptor+ (LepR+) BM stromal cells. To clarify how Bmi1 regulates the HSC niche, we specifically deleted Bmi1 in LepR+ cells in mice. The Bmi1 deletion promoted the adipogenic differentiation of LepR+ stromal cells and caused progressive fatty changes in the BM of limb bones with age, resulting in reductions in the numbers of HSCs and progenitors in BM and enhanced extramedullary hematopoiesis. This adipogenic change was also evident during BM regeneration after irradiation. Several adipogenic regulator genes appeared to be regulated by Bmi1. Our results indicate that Bmi1 keeps the adipogenic differentiation program repressed in BM stromal cells to maintain the integrity of the HSC niche.

CBX2 is required to stabilize the testis pathway by repressing Wnt signaling.

XX and XY fetal gonads are initially bipotential, poised between the ovary and testis fate. Multiple lines of evidence suggest that commitment to testis fate requires the repression of genes associated with ovary fate. It was previously shown that loss of CBX2, the subunit of the Polycomb Repressive Complex 1 (PRC1) that binds H3K27me3 and mediates silencing, leads to ovary development in XY mice and humans. While it had been proposed that CBX2 is an activator of the testis-determining gene Sry, we investigated the alternative possibility that CBX2 has a direct role as a repressor of the antagonistic ovary-promoting pathway. To investigate this possibility, we developed a quantitative genome-wide profile of the repressive histone mark H3K27me3 and its active counterpart H3K4me3 in isolated XY and XX gonadal supporting cells before and after sex determination. We show that testis and ovary sex-determining (SD) genes are bivalent before sex determination, providing insight into how the bipotential state of the gonad is established at the epigenetic level. After sex determination, many SD genes of the alternate pathway remain bivalent, possibly contributing to the ability of these cells to transdifferentiate even in adults. The finding that many genes in the Wnt signaling pathway were targeted for H3K27me3-mediated repression in Sertoli cells led us to test whether deletion of Wnt4 could rescue testis development in Cbx2 mutants. We show that Sry expression and testis development were rescued in XY Cbx2-/-;Wnt4-/- mice. Furthermore, we show that CBX2 directly binds the downstream Wnt signaler Lef1, an ovary-promoting gene that remains bivalent in Sertoli cells. Our results suggest that stabilization of the testis fate requires CBX2-mediated repression of bivalent ovary-determining genes, which would otherwise block testis development.

Knockdown of RNF2 enhances the radiosensitivity of squamous cell carcinoma in lung.

A previous study has reported that knockdown of RING finger protein 2 (RNF2) increases the radiosensitivity of esophageal cancer cells both in vitro and in vivo. However, the effect of RNF2 knockdown on radiosensitivity in squamous cell carcinoma (SqCC) remains unknown. For this, NCI-H226 and SK-MES-1 cells were exposed to X-ray irradiation and then RNF2 levels were determined. RNF2 was knocked-down and stable transfectants were selected. Radiosensitivity, cell proliferation, apoptosis, cell cycle, and γ-H2AX foci formation were evaluated. Interaction among ataxia telangiectasia mutated protein (ATM), mediator of DNA damage checkpoint 1 (MDC1), and H2AX were examined. Xenograft models were used to explore the effect of RNF2 knockdown on radiosensitivity in vivo. The results showed that RNF2 expression was significantly increased by X-ray irradiation. RNF2 knockdown combined with X-ray irradiation markedly inhibited cell proliferation, caused cell cycle arrest at the G1 phase, and induced cell apoptosis. In addition, RNF2 knockdown enhanced the radiosensitivity of SqCC cells, inhibited irradiation-induced γ-H2AX foci formation, and impaired the interactions among ATM, MDC1, and H2AX. Furthermore, combination of RNF2 knockdown and X-ray irradiation suppressed tumor growth and promoted tumor cell apoptosis in vivo. RNF2 may be a new therapeutic target to enhance the radiosensitivity of SqCC cells in lung.

Impaired Expression of Rearranged Immunoglobulin Genes and Premature p53 Activation Block B Cell Development in BMI1 Null Mice.

B cell development is a highly regulated process that requires stepwise rearrangement of immunoglobulin genes to generate a functional B cell receptor (BCR). The polycomb group protein BMI1 is required for B cell development, but its function in developing B cells remains poorly defined. We demonstrate that BMI1 functions in a cell-autonomous manner at two stages during early B cell development. First, loss of BMI1 results in a differentiation block at the pro-B cell to pre-B cell transition due to the inability of BMI1-deficient cells to transcribe newly rearranged Igh genes. Accordingly, introduction of a pre-rearranged Igh allele partially restored B cell development in Bmi1-/- mice. In addition, BMI1 is required to prevent premature p53 signaling, and as a consequence, Bmi1-/- large pre-B cells fail to properly proliferate. Altogether, our results clarify the role of BMI1 in early B cell development and uncover an unexpected function of BMI1 during VDJ recombination.

Effect of CBX7 deficiency on the socket healing after tooth extractions.

CBX7 is shown to down-regulate the expression of osteopontin (OPN) that is associated with osteoblast function. Here, we studied the role of CBX7 in the wound healing of tooth extraction socket in which osteoblast activity is critical via comparison between CBX7-knockout (CBX7-/-) mice and their wild-type (WT) counterparts of 6 weeks old with maxillary first molar extracted. Mice were euthanized at 7, 14, and 21 days after extractions, and alveolar sockets were assessed by semi-quantitative histomorphometry for hard tissue healing, including new bone fill (Masson's trichrome staining), osteoblast activity (OPN/osterix, Osx), osteoclast activity (tartrate-resistant acid phosphatase, TRAP), and for soft tissue healing, including blood vessels (alpha smooth muscle actin, α-SMA). Also, the bone microarchitecture was evaluated by micro-CT. In radiological analysis, CBX7-/- mice increased bone mass significantly more than WT mice did. Consistently, both the amount of new bone fill and OPN/Osx-immunopositive cells in the extraction sockets were significantly increased in CBX7-/- mice at each time point with respect to their WT siblings, while osteoclast number exhibited a trend of more increase in CBX7-/- mice at all time points as well. In agreement with enhanced bone formation during socket healing, significantly elevated α-SMA-immunopositive area was noted in CBX7-/- mice in contrast to WT mice. Taken together, these data suggest that CBX7 deficiency has a positive effect on tooth extraction socket healing.

Modeling Late-Onset Sporadic Alzheimer's Disease through BMI1 Deficiency.

Late-onset sporadic Alzheimer's disease (AD) is the most prevalent form of dementia, but its origin remains poorly understood. The Bmi1/Ring1 protein complex maintains transcriptional repression of developmental genes through histone H2A mono-ubiquitination, and Bmi1 deficiency in mice results in growth retardation, progeria, and neurodegeneration. Here, we demonstrate that BMI1 is silenced in AD brains, but not in those with early-onset familial AD, frontotemporal dementia, or Lewy body dementia. BMI1 expression was also reduced in cortical neurons from AD patient-derived induced pluripotent stem cells but not in neurons overexpressing mutant APP and PSEN1. BMI1 knockout in human post-mitotic neurons resulted in amyloid beta peptide secretion and deposition, p-Tau accumulation, and neurodegeneration. Mechanistically, BMI1 was required to repress microtubule associated protein tau (MAPT) transcription and prevent GSK3beta and p53 stabilization, which otherwise resulted in neurodegeneration. Restoration of BMI1 activity through genetic or pharmaceutical approaches could represent a therapeutic strategy against AD.

PRC1 Fine-tunes Gene Repression and Activation to Safeguard Skin Development and Stem Cell Specification.

Polycomb repressive complexes (PRCs) 1 and 2 are essential chromatin regulators of cell identity. PRC1, a dominant executer of Polycomb-mediated control, functions as multiple sub-complexes that possess catalytic-dependent H2AK119 mono-ubiquitination (H2AK119ub) and catalytic-independent activities. Here, we show that, despite its well-established repressor functions, PRC1 binds to both silent and active genes. Through in vivo loss-of-function studies, we show that global PRC1 function is essential for skin development and stem cell (SC) specification, whereas PRC1 catalytic activity is dispensable. Further dissection demonstrated that both canonical and non-canonical PRC1 complexes bind to repressed genes, marked by H2AK119ub and PRC2-mediated H3K27me3. Interestingly, loss of canonical PRC1, PRC1 catalytic activity, or PRC2 leads to expansion of mechanosensitive Merkel cells in neonatal skin. Non-canonical PRC1 complexes, however, also bind to and promote expression of genes critical for skin development and SC formation. Together, our findings highlight PRC1's diverse roles in executing a precise developmental program.

The Evaluation of BMI1 Posttranslational Modifications During Retinal Degeneration to Understand BMI1 Action on Photoreceptor Death Execution.

Retinitis Pigmentosa (RP) is a class of hereditary retinal dystrophy associated with gradual visual failure and a subsequent loss of light-sensitive cells in the retina, leading to blindness. Many mutated genes were found to be causative of this disease. Despite a number of compiling efforts, the process of cell death in photoreceptors remains to be clearly elucidated. We recently reported an abnormal cell cycle reentry in photoreceptors undergoing degeneration in Rd1 mice, a model of RP, and identified the polycomb repressive complex 1 (PRC1) core component BMI1 as a critical molecular factor orchestrating the cell death mechanism. As the cell death rescue in Rd1;Bmi-1 KO mice was independent on the conventional Ink4a/Arf pathways, we now explored the structural properties of BMI1 in order to examine the differential expression of its posttranslational modifications in Rd1 retina. Our results suggest that BMI1 cell death induction in Rd1 is not related to its phosphorylation status. We therefore propose the epigenetic activity of BMI1 as an alternative route for BMI1-mediated toxicity in Rd1.

Modulation of hippocampal synapse maturation by activity-regulated E3 ligase via non-canonical pathway.

Development of functional synapses is crucial for the transmission and storage of information in the brain. Post establishment of the initial synaptic contact, synapses are stabilized through neuronal activity-induced signals. Emerging studies have implicated ubiquitination; a reversible posttranslational modification, as a key regulatory switch that modulates synapse development through proteasomal degradation. Ubiquitination of proteins is precisely regulated by E3 ligases, a set of enzymes that bind to specific substrates to facilitate the conjugation of monomeric or polymeric ubiquitin. However, the identity of specific E3 ubiquitin ligases that influence activity-dependent maturation of synapses and the mechanism by which ubiquitination of proteins regulate functional synapse development remain elusive. Here, we have identified a RING domain containing E3 ligase, Rnf2, asan activity-regulated factor that modulates glutamatergic synapse development in the hippocampus. Rnf2 is a synapse associated E3 ligase that is stabilized by neuronal activity through self-polyubiquitination. We have shown that neuronal activity shifts the balance toward stabilization of Rnf2 through self-polyubiquitination rather than triggering its degradation through polyubiquitination by Ube3A, an E3 ligase implicated in Angelman Syndrome. Our synapse density measurements and whole-cell patch-clamp recordings have revealed that the loss of Rnf2 function in cultured hippocampal neurons result in the development of 'silent' synapses that lack GluA1 containing functional AMPA receptors. These results provide a plausible mechanistic approach toward understanding how synapse maturation is regulated via the activity-dependent stabilization of Rnf2 through a non-canonical function of polyubiquitination.

BMI1 and MEL18 Promote Colitis-Associated Cancer in Mice via REG3B and STAT3.

BACKGROUND & AIMS: Polycomb group proteins are epigenetic factors that silence gene expression; they are dysregulated in cancer cells and contribute to carcinogenesis by unclear mechanisms. We investigated whether BMI1 proto-oncogene, polycomb ring finger (BMI1), and polycomb group ring finger 2 (PCGF2, also called MEL18) are involved in the initiation and progression of colitis-associated cancer (CAC) in mice. METHODS: We generated mice containing floxed alleles of Bmi1 and/or Mel18 and/or Reg3b using the villin-Cre promoter (called Bmi1ΔIEC, Mel18ΔIEC, DKO, and TKO mice). We also disrupted Bmi1 and/or Mel18 specifically in intestinal epithelial cells (IECs) using the villin-CreERT2-inducible promoter. CAC was induced in cre-negative littermate mice (control) and mice with conditional disruption of Bmi1 and/or Mel18 by intraperitoneal injection of azoxymethane (AOM) followed by addition of dextran sulfate sodium (DSS) to drinking water. Colon tissues were collected from mice and analyzed by histology and immunoblots; IECs were isolated and used in cDNA microarray analyses. RESULTS: Following administration of AOM and DSS, DKO mice developed significantly fewer polyps than control, Bmi1ΔIEC, Mel18ΔIEC, Reg3bΔIEC, or TKO mice. Adenomas in the colons of DKO mice were low-grade dysplasias, whereas adenomas in control, Bmi1ΔIEC, Mel18ΔIEC, Reg3bΔIEC, or TKO mice were high-grade dysplasias with aggressive invasion of the muscularis mucosa. Disruption of Bmi1 and Mel18 (DKO mice) during late stages of carcinogenesis significantly reduced the numbers of large adenomas and the load of total adenomas, reduced proliferation, and increased apoptosis in colon tissues. IECs isolated from DKO mice after AOM and DSS administration had increased expression of Reg3b compared with control, Bmi1ΔIEC, or Mel18ΔIEC mice. Expression of REG3B was sufficient to inhibit cytokine-induced activation of STAT3 in IECs. The human REG3ß protein, the functional counterpart of mouse REG3B, inhibited STAT3 activity in human 293T cells, and its expression level in colorectal tumors correlated inversely with pSTAT3 level and survival times of patients. CONCLUSIONS: BMI1 and MEL18 contribute to the development of CAC in mice by promoting proliferation and reducing apoptosis via suppressing expression of Reg3b. REG3B negatively regulates cytokine-induced activation of STAT3 in colon epithelial cells. This pathway might be targeted in patients with colitis to reduce carcinogenesis.

P16 INK4a Deletion Ameliorated Renal Tubulointerstitial Injury in a Stress-induced Premature Senescence Model of Bmi-1 Deficiency.

To determine whether p16 INK4a deletion ameliorated renal tubulointerstitial injury by inhibiting a senescence-associated secretory phenotype (SASP) in Bmi-1-deficient (Bmi-1 -/-) mice, renal phenotypes were compared among 5-week-old Bmi-1 and p16 INK4a double-knockout, and Bmi-1 -/- and wild-type mice. Fifth-passage renal interstitial fibroblasts (RIFs) from the three groups were analyzed for senescence and proliferation. The effect of Bmi-1 deficiency on epithelial-to-mesenchymal transition (EMT) was examined in Bmi-1-knockdown human renal proximal tubular epithelial (HK2) cells, which were treated with concentrated conditioned medium (CM) from the fifth-passage renal interstitial fibroblasts (RIFs) of above three group mice or with exogenous TGF-ß1. Our results demonstrated that p16 INK4a deletion largely rescued renal aging phenotypes caused by Bmi-1 deficiency, including impaired renal structure and function, decreased proliferation, increased apoptosis, senescence and SASP, DNA damage, NF-κB and TGF-ß1/Smad signal activation, inflammatory cell infiltration, and tubulointerstitial fibrosis and tubular atrophy. P16 INK4a deletion also promoted proliferation, reduced senescence and SASP of RIFs and subsequently inhibited EMT of Bmi-1-knockdown HK2 cells. TGF-ß1 further induced the EMT of Bmi-1-knockdown HK2 cells. Thus, p16 INK4a positive senescent cells would be a therapeutic target for preventing renal tubulointerstitial injury.

Hypothalamic stem cells control ageing speed partly through exosomal miRNAs.

It has been proposed that the hypothalamus helps to control ageing, but the mechanisms responsible remain unclear. Here we develop several mouse models in which hypothalamic stem/progenitor cells that co-express Sox2 and Bmi1 are ablated, as we observed that ageing in mice started with a substantial loss of these hypothalamic cells. Each mouse model consistently displayed acceleration of ageing-like physiological changes or a shortened lifespan. Conversely, ageing retardation and lifespan extension were achieved in mid-aged mice that were locally implanted with healthy hypothalamic stem/progenitor cells that had been genetically engineered to survive in the ageing-related hypothalamic inflammatory microenvironment. Mechanistically, hypothalamic stem/progenitor cells contributed greatly to exosomal microRNAs (miRNAs) in the cerebrospinal fluid, and these exosomal miRNAs declined during ageing, whereas central treatment with healthy hypothalamic stem/progenitor cell-secreted exosomes led to the slowing of ageing. In conclusion, ageing speed is substantially controlled by hypothalamic stem cells, partially through the release of exosomal miRNAs.

Loss of Polycomb Group Protein Pcgf1 Severely Compromises Proper Differentiation of Embryonic Stem Cells.

The Polycomb repressive complex 1 (PRC1) is essential for fate decisions of embryonic stem (ES) cells. Emerging evidence suggests that six major variants of PRC1 complex, defined by the mutually exclusive presence of Pcgf subunit, regulate distinct biological processes, yet very little is known about the mechanism by which each version of PRC1 instructs and maintains cell fate. Here, we disrupted the Pcgf1, also known as Nspc1 and one of six Pcgf paralogs, in mouse ES cells by the CRISPR/Cas9 technology. We showed that although these mutant cells were viable and retained normal self-renewal, they displayed severe defects in differentiation in vitro. To gain a better understanding of the role of Pcgf1 in transcriptional control of differentiation, we analysed mRNA profiles from Pcgf1 deficient cells using RNA-seq. Interestingly, we found that Pcgf1 positively regulated expression of essential transcription factors involved in ectoderm and mesoderm differentiation, revealing an unexpected function of Pcgf1 in gene activation during ES cell lineage specification. Chromatin immunoprecipitation experiments demonstrated that Pcgf1 deletion caused a decrease in Ring1B and its associated H2AK119ub1 mark binding to target genes. Altogether, our results suggested an unexpected function of Pcgf1 in gene activation during ES cell maintenance.

Bmi1 Loss in the Organ of Corti Results in p16ink4a Upregulation and Reduced Cell Proliferation of Otic Progenitors In Vitro.

The mature mammalian organ of Corti does not regenerate spontaneously after injury, mainly due to the absence of cell proliferation and the depletion of otic progenitors with age. The polycomb gene B lymphoma Mo-MLV insertion region 1 homolog (Bmi1) promotes proliferation and cell cycle progression in several stem cell populations. The cell cycle inhibitor p16ink4a has been previously identified as a downstream target of Bmi1. In this study, we show that Bmi1 is expressed in the developing inner ear. In the organ of Corti, Bmi1 expression is temporally regulated during embryonic and postnatal development. In contrast, p16ink4a expression is not detectable during the same period. Bmi1-deficient mice were used to investigate the role of Bmi1 in cochlear development and otosphere generation. In the absence of Bmi1, the postnatal organ of Corti displayed normal morphology at least until the end of the first postnatal week, suggesting that Bmi1 is not required for the embryonic or early postnatal development of the organ of Corti. However, Bmi1 loss resulted in the reduced sphere-forming capacity of the organ of Corti, accompanied by the decreased cell proliferation of otic progenitors in otosphere cultures. This reduced proliferative capacity was associated with the upregulation of p16ink4a in vitro. Viral vector-mediated overexpression of p16ink4a in wildtype otosphere cultures significantly reduced the number of generated otospheres in vitro. The findings strongly suggest a role for Bmi1 as a promoter of cell proliferation in otic progenitor cells, potentially through the repression of p16ink4a.

CBX7 deficiency plays a positive role in dentin and alveolar bone development.

To clarify the role of CBX7 deficiency in dentin and alveolar bone development, the dental and mandibular phenotypes of homozygous CBX7-knockout (CBX7(-/-)) mice were compared with their wild-type (WT) counterparts at 3 weeks age. In contrast to WT littermates, dental volume and dentin sialoprotein-positive area were significantly increased, whereas the area ratio of predentin to dentin was decreased markedly in CBX7(-/-) mice. Mineral density, cortical thickness, alveolar bone volume, type I collagen and osterix-immunopositive area, osteoblast number and activity, protein expression and mRNA level of Runt-related transcription factor 2 (Runx2), alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2 (BMP2) were all remarkably increased, while osteoclast number and activity, and mRNA expression ratio of NF-κB ligand (RANKL) to osteoprotegerin (opg) were all decreased significantly in the alveolar bone of CBX7(-/-) mice compared with their WT counterparts. Moreover, proliferating cell nuclear antigen (PCNA)-positive cells were found more in Hertwig' s epithelial root sheath of CBX7(-/-) mice, and their protein level of cyclin E1, cyclin-dependent kinase 2 (CDK2) were correspondingly increased in contrast to WT mice. Taken together, these results of this study suggest that CBX7 deficiency plays a positive role in dentin and alveolar bone formation.

The Polycomb Repressive Complex 1 Protein BMI1 Is Required for Constitutive Heterochromatin Formation and Silencing in Mammalian Somatic Cells.

The polycomb repressive complex 1 (PRC1), containing the core BMI1 and RING1A/B proteins, mono-ubiquitinylates histone H2A (H2A(ub)) and is associated with silenced developmental genes at facultative heterochromatin. It is, however, assumed that the PRC1 is excluded from constitutive heterochromatin in somatic cells based on work performed on mouse embryonic stem cells and oocytes. We show here that BMI1 is required for constitutive heterochromatin formation and silencing in human and mouse somatic cells. BMI1 was highly enriched at intergenic and pericentric heterochromatin, co-immunoprecipitated with the architectural heterochromatin proteins HP1, DEK1, and ATRx, and was required for their localization. In contrast, BRCA1 localization was BMI1-independent and partially redundant with that of BMI1 for H2A(ub) deposition, constitutive heterochromatin formation, and silencing. These observations suggest a dynamic and developmentally regulated model of PRC1 occupancy at constitutive heterochromatin, and where BMI1 function in somatic cells is to stabilize the repetitive genome.