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  • Cyclin D1 fine-tunes the neurogenic output of embryonic retinal progenitor cells. 19416500

    Maintaining the correct balance of proliferation versus differentiation in retinal progenitor cells (RPCs) is essential for proper development of the retina. The cell cycle regulator cyclin D1 is expressed in RPCs, and mice with a targeted null allele at the cyclin D1 locus (Ccnd1-/-) have microphthalmia and hypocellular retinas, the latter phenotype attributed to reduced RPC proliferation and increased photoreceptor cell death during the postnatal period. How cyclin D1 influences RPC behavior, especially during the embryonic period, is unclear.In this study, we show that embryonic RPCs lacking cyclin D1 progress through the cell cycle at a slower rate and exit the cell cycle at a faster rate. Consistent with enhanced cell cycle exit, the relative proportions of cell types born in the embryonic period, such as retinal ganglion cells and photoreceptor cells, are increased. Unexpectedly, cyclin D1 deficiency decreases the proportions of other early born retinal neurons, namely horizontal cells and specific amacrine cell types. We also found that the laminar positioning of horizontal cells and other cell types is altered in the absence of cyclin D1. Genetically replacing cyclin D1 with cyclin D2 is not efficient at correcting the phenotypes due to the cyclin D1 deficiency, which suggests the D-cyclins are not fully redundant. Replacement with cyclin E or inactivation of cyclin-dependent kinase inhibitor p27Kip1 restores the balance of RPCs and retinal cell types to more normal distributions, which suggests that regulation of the retinoblastoma pathway is an important function for cyclin D1 during embryonic retinal development.Our findings show that cyclin D1 has important roles in RPC cell cycle regulation and retinal histogenesis. The reduction in the RPC population due to a longer cell cycle time and to an enhanced rate of cell cycle exit are likely to be the primary factors driving retinal hypocellularity and altered output of precursor populations in the embryonic Ccnd1-/- retina.
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  • Biomechanical stimulation of osteoblast gene expression requires phosphorylation of the RUNX2 transcription factor. 22337141

    Bone can adapt its structure in response to mechanical stimuli. At the cellular level, this involves changes in chromatin organization, gene expression, and differentiation, but the underlying mechanisms are poorly understood. Here we report on the involvement of RUNX2, a bone-related transcription factor, in this process. Fluid flow shear stress loading of preosteoblasts stimulated translocation of extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) to the nucleus where it phosphorylated RUNX2 on the chromatin of target genes, and increased histone acetylation and gene expression. MAPK signaling and two RUNX2 phosphoacceptor sites, S301 and S319, were critical for this response. Similarly, in vivo loading of mouse ulnae dramatically increased ERK and RUNX2 phosphorylation as well as expression of osteoblast-related genes. These findings establish ERK/MAPK-mediated phosphorylation of RUNX2 as a critical step in the response of preosteoblasts to dynamic loading and define a novel mechanism to explain how mechanical signals induce gene expression in bone.
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  • Dynamic interplay of transcriptional machinery and chromatin regulates "late" expression of the chemokine RANTES in T lymphocytes. 17074812

    The chemokine RANTES (regulated upon activation normal T cell expressed and secreted) is expressed "late" (3 to 5 days) after activation in T lymphocytes. In order to understand the molecular events that accompany changes in gene expression, a detailed analysis of the interplay between transcriptional machinery and chromatin on the RANTES promoter over time was undertaken. Krüppel-like factor 13 (KLF13), a sequence-specific DNA binding transcription factor, orchestrates the induction of RANTES expression in T lymphocytes by ordered recruitment of effector molecules, including Nemo-like kinase, p300/cyclic AMP response element binding protein (CBP), p300/CBP-associated factor, and Brahma-related gene 1, that initiate sequential changes in phosphorylation and acetylation of histones and ATP-dependent chromatin remodeling near the TATA box of the RANTES promoter. These events recruit RNA polymerase II to the RANTES promoter and are responsible for late expression of RANTES in T lymphocytes. Therefore, KLF13 is a key regulator of late RANTES expression in T lymphocytes.
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  • BMP-induced REST regulates the establishment and maintenance of astrocytic identity. 20351065

    Once they have differentiated, cells retain their individual character and repress genes that are specifically expressed in other cell lineages, but how alternative fate choice is restricted during and/or after differentiation remains unclear. In the mammalian central nervous system, neurons, astrocytes, and oligodendrocytes are generated throughout life from common tripotent neural progenitor cells (NPCs). Bone morphogenetic proteins (BMPs) are well-known astrocyte-inducing cytokines. We show here that the expression of a transcriptional repressor, RE1 silencer of transcription (REST)/neuron-restrictive silencer factor (NRSF), is up-regulated and sustained by BMP signal activation in the course of astrocytic differentiation of NPCs, and restricts neuronal differentiation. We further show that, in differentiated astrocytes, endogenous REST/NRSF associates with various neuronal genes and that disruption of its function resulted in their derepression, thereby explaining how ectopic neuronal gene expression is prevented in cells with astrocytic traits. Collectively, our results suggest that REST/NRSF functions as a molecular regulator of the nonneuronal phenotype in astrocytes.
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  • Deregulated E2f-2 underlies cell cycle and maturation defects in retinoblastoma null erythroblasts. 17923680

    By assessing the contribution of deregulated E2F activity to erythroid defects in Rb null mice, we have identified E2f-2 as being upregulated in end-stage red cells, where we show it is the major pRb-associated E2f and the predominant E2f detected at key target gene promoters. Consistent with its expression pattern, E2f-2 loss restored terminal erythroid maturation to Rb null red cells, including the ability to undergo enucleation. Deletion of E2f-2 also extended the life span of Rb null mice despite persistent defects in placental development, indicating that deregulated E2f-2 activity in differentiating erythroblasts contributes to the premature lethality of Rb null mice. We show that the aberrant entry of Rb null erythroblasts into S phase at times in differentiation when wild-type erythroblasts are exiting the cell cycle is inhibited by E2f-2 deletion. E2f-2 loss induced cell cycle arrest in both wild-type and Rb null erythroblasts and was associated with increased DNA double-strand breaks. These results implicate deregulated E2f-2 in the cell cycle defects observed in Rb null erythroblasts and reveal a novel role for E2f-2 during terminal red blood cell differentiation. The identification of a tissue-restricted role for E2f-2 in erythropoiesis highlights the nonredundant nature of E2f transcription factor activities in cell growth and differentiation.
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  • Maize histone deacetylase hda101 is involved in plant development, gene transcription, and sequence-specific modulation of histone modification of genes and repeats. 17468264

    Enzymes catalyzing histone acetylation and deacetylation contribute to the modulation of chromatin structure, thus playing an important role in regulating gene and genome activity. We showed that downregulation and overexpression of the maize (Zea mays) Rpd3-type hda101 histone deacetylase gene induced morphological and developmental defects. Total levels of acetylated histones and histone acetylation of both repetitive and nonrepetitive sequences were affected in hda101 transgenic mutants. However, only transcript levels of genes but not repeats were altered. In particular, hda101 transgenic mutants showed differential expression of genes involved in vegetative-to-reproductive transition, such as liguleless2 and knotted-like genes and their repressor rough sheath2, which are required for meristem initiation and maintenance. Perturbation of hda101 expression also affected histone modifications other than acetylation, including histone H3 dimethylation at Lys-4 and Lys-9 and phosphorylation at Ser-10. Our results indicate that hda101 affects gene transcription and provide evidence of its involvement in setting the histone code, thus mediating developmental programs. Possible functional differences between maize hda101 and its Arabidopsis thaliana ortholog HDA19 are discussed.
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  • Defective survival of proliferating Sertoli cells and androgen receptor function in a mouse model of the ATR-X syndrome. 21427128

    X-linked ATR-X (alpha thalassemia, mental retardation, X-linked) syndrome in males is characterized by mental retardation, facial dysmorphism, alpha thalassemia and urogenital abnormalities, including small testes. It is unclear how mutations in the chromatin-remodeling protein ATRX cause these highly specific clinical features, since ATRX is widely expressed during organ development. To investigate the mechanisms underlying the testicular defects observed in ATR-X syndrome, we generated ScAtrxKO (Sertoli cell Atrx knockout) mice with Atrx specifically inactivated in the supporting cell lineage (Sertoli cells) of the mouse testis. ScAtrxKO mice developed small testes and discontinuous tubules, due to prolonged G2/M phase and apoptosis of proliferating Sertoli cells during fetal life. Apoptosis might be a consequence of the cell cycle defect. We also found that the onset of spermatogenesis was delayed in postnatal mice, with a range of spermatogenesis defects evident in adult ScAtrxKO mice. ATRX and the androgen receptor (AR) physically interact in the testis and in the Sertoli cell line TM4 and co-operatively activate the promoter of Rhox5, an important direct AR target. We also demonstrate that ATRX directly binds to the Rhox5 promoter in TM4 cells. Finally, gene expression of Rhox5 and of another AR-dependent gene, Spinlw1, was reduced in ScAtrxKO testes. These data suggest that ATRX can directly enhance the expression of androgen-dependent genes through physical interaction with AR. Recruitment of ATRX by DNA sequence-specific transcription factors could be a general mechanism by which ATRX achieves tissue-specific transcriptional regulation which could explain the highly specific clinical features of ATR-X syndrome when ATRX is mutated.
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  • Increased expression of FoxM1 transcription factor in respiratory epithelium inhibits lung sacculation and causes Clara cell hyperplasia. 20816795

    Foxm1 is a member of the Forkhead Box (Fox) family of transcription factors. Foxm1 (previously called Foxm1b, HFH-11B, Trident, Win, or MPP2) is expressed in multiple cell types and plays important roles in cellular proliferation, differentiation and tumorigenesis. Genetic deletion of Foxm1 from mouse respiratory epithelium during initial stages of lung development inhibits lung maturation and causes respiratory failure after birth. However, the role of Foxm1 during postnatal lung morphogenesis remains unknown. In the present study, Foxm1 expression was detected in epithelial cells of conducting and peripheral airways and changing dynamically with lung maturation. To discern the biological role of Foxm1 in the prenatal and postnatal lung, a novel transgenic mouse line that expresses a constitutively active form of FoxM1 (FoxM1 N-terminal deletion mutant or FoxM1-ΔN) under the control of lung epithelial-specific SPC promoter was produced. Expression of the FoxM1-ΔN transgene during embryogenesis caused epithelial hyperplasia, inhibited lung sacculation and expression of the type II epithelial marker, pro-SPC. Expression of FoxM1-ΔN mutant during the postnatal period did not influence alveologenesis but caused focal airway hyperplasia and increased proliferation of Clara cells. Likewise, expression of FoxM1-ΔN mutant in conducting airways with Scgb1a1 promoter was sufficient to induce Clara cell hyperplasia. Furthermore, FoxM1-ΔN cooperated with activated K-Ras to induce lung tumor growth in vivo. Increased activity of Foxm1 altered lung sacculation, induced proliferation in the respiratory epithelium and accelerated lung tumor growth, indicating that precise regulation of Foxm1 is critical for normal lung morphogenesis and development of lung cancer.
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  • Human cells enter mitosis with damaged DNA after treatment with pharmacological concentrations of genotoxic agents. 22686412

    In the present paper, we report that mitosis is a key step in the cellular response to genotoxic agents in human cells. Cells with damaged DNA recruit γH2AX (phosphorylated histone H2AX), phosphorylate Chk1 (checkpoint kinase 1) and arrest in the G2-phase of the cell cycle. Strikingly, nearly all cells escape the DNA damage checkpoint and become rounded, by a mechanism that correlates with Chk1 dephosphorylation. The rounded cells are alive and in mitosis as measured by low phospho-Tyr(15) Cdk1 (cyclin-dependent kinase 1), high Cdk activity, active Plk1 (Polo-like kinase 1) and high phospho-histone H3 signals. This phenomenon is independent of the type of DNA damage, but is dependent on pharmacologically relevant doses of genotoxicity. Entry into mitosis is likely to be caused by checkpoint adaptation, and the HT-29 cell-based model provides a powerful experimental system in which to explore its molecular basis. We propose that mitosis with damaged DNA is a biologically significant event because it may cause genomic rearrangement in cells that survive genotoxic damage.
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  • Substrate degradation by the anaphase promoting complex occurs during mitotic slippage. 20436289

    Microtubule targeting drugs are successful in chemotherapy because they indefinitely activate the spindle assembly checkpoint. The spindle assembly checkpoint monitors proper attachment of all kinetochores to microtubules and tension between the kinetochores of sister chromatids to prevent premature anaphase entry. To this end, the activated spindle assembly checkpoint suppresses the E3 ubiquitin ligase activity of the anaphase-promoting complex (APC). In the continued presence of conditions that activate the spindle assembly checkpoint, cells eventually escape from mitosis by "slippage". It has not been directly tested whether APC activation accompanies slippage. Using cells blocked in mitosis with the microtubule assembly inhibitor nocodazole, we show that mitotic APC substrates are degraded upon mitotic slippage. To confirm that APC is normally activated upon mitotic slippage we have found that knockdown of Cdc20 and Cdh1, two mitotic activators of APC, prevents the degradation of APC substrates during mitotic slippage. We provide the first direct demonstration that despite conditions that activate the spindle checkpoint, APC is indeed activated upon mitotic slippage of cells to interphase cells. Activation of the spindle checkpoint by microtubule targeting drugs used in chemotherapy may not indefinitely prevent APC activation.
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