Cell proliferation and differentiation are highly coordinated during normal development. Many tumor cells exhibit both uncontrolled proliferation and a block to terminal differentiation. To ...understand the mechanisms coordinating these two processes, we have investigated the relation between cyclin-dependent kinase (CDK) activities and the block to differentiation in murine erythroleukemia (MEL) cells. We found that CDK6 (but not CDK4) is rapidly downregulated as MEL cells are induced to re-enter erythroid differentiation and that maintenance of CDK6 (but not CDK4) activity by transfection blocks differentiation. Moreover, we found that PU.1, an Ets transcription factor that is oncogenic in erythroid cells and also can block their differentiation, controls the synthesis of CDK6 mRNA. These results suggest a mechanism for coupling proliferation and the block to differentiation in these leukemic cells through the action of an oncogenic transcription factor (PU.1) on a key cell cycle regulator (CDK6). Our findings suggest that studying the relative roles of CDK6 and CDK4 in other types of malignant cells will be important in designing approaches for cell cycle inhibition and differentiation therapy in cancer.
Sleep and wakefulness are regulated primarily by inhibitory interactions between the hypothalamus and brainstem. The expression of the states of rapid eye movement (REM) sleep and non-REM (NREM) ...sleep also are correlated with the activity of groups of REM-off and REM-on neurons in the dorsal brainstem. However, the contribution of ventral brainstem nuclei to sleep regulation has been little characterized to date. Here we examined sleep and wakefulness in mice deficient in a homeobox transcription factor, Goosecoid-like (Gscl), which is one of the genes deleted in DiGeorge syndrome or 22q11 deletion syndrome. The expression of Gscl is restricted to the interpeduncular nucleus (IP) in the ventral region of the midbrain–hindbrain transition. The IP has reciprocal connections with several cell groups implicated in sleep/wakefulness regulation. Although Gscl -/- mice have apparently normal anatomy and connections of the IP, they exhibited a reduced total time spent in REM sleep and fewer REM sleep episodes. In addition, Gscl -/- mice showed reduced theta power during REM sleep and increased arousability during REM sleep. Gscl -/- mice also lacked the expression of DiGeorge syndrome critical region 14 (Dgcr14) in the IP. These results indicate that the absence of Gscl and Dgcr14 in the IP results in altered regulation of REM sleep.
Within the nucleus of eukaryotic cells, chromatin is organized into compact, silent regions called heterochromatin and more loosely packaged regions of euchromatin where transcription is more active. ...Although the existence of heterochromatin has been known for many years, the cellular factors responsible for its formation have only recently been identified. Two key factors involved in heterochromatin formation in Drosophila are the H3 lysine 9 methyltransferase Su(var)3-9 and heterochromatin protein 1 (HP1). The linker histone H1 also plays a major role in heterochromatin formation in Drosophila by interacting with Su(var)3-9 and helping to recruit it to heterochromatin. Drosophila STAT (Signal transducer and activator of transcription) (STAT92E) has also been shown to be involved in the maintenance of heterochromatin, but its relationship to the H1-Su(var)3-9 heterochromatin pathway is unknown. STAT92E is also involved in tumor formation in flies. Hyperactive Janus kinase (JAK)-STAT signaling due to a mutation in Drosophila JAK (Hopscotch) causes hematopoietic tumors.
We show here that STAT92E is a second partner of H1 in the regulation of heterochromatin structure. H1 physically interacts with STAT92E and regulates its ectopic localization in the chromatin. Mis-localization of STAT92E due to its hyperphosphorylation or H1 depletion disrupts heterochromatin integrity. The contribution of the H1-STAT pathway to heterochromatin formation is mechanistically distinct from that of H1 and Su(var)3-9. The recruitment of STAT92E to chromatin by H1 also plays an important regulatory role in JAK-STAT induced tumors in flies. Depleting the linker histone H1 in flies carrying the oncogenic hopscotch (Tum-l) allele enhances tumorigenesis, and H1 overexpression suppresses tumorigenesis.
Our results suggest the existence of two independent pathways for heterochromatin formation in Drosophila, one involving Su(var)3-9 and HP1 and the other involving STAT92E and HP1. The H1 linker histone directs both pathways through physical interactions with Su(var)3-9 and STAT92E, as well with HP1. The physical interaction of H1 and STAT92E confers a regulatory role on H1 in JAK-STAT signaling. H1 serves as a molecular reservoir for STAT92E in chromatin, enabling H1 to act as a tumor suppressor and oppose an oncogenic mutation in the JAK-STAT signaling pathway.
Malignant transformation often leads to both loss of normal proliferation control and inhibition of cell differentiation.
Some tumor cells can be stimulated to reenter their differentiation program ...and to undergo terminal growth arrest. The in vitro differentiation of mouse erythroleukemia (MEL) cells is an important example of tumor cell reprogramming. MEL cells are malignant
erythroblasts that are blocked from differentiating into mature RBC due to dysregulated expression of the transcription factor
PU.1, which binds to and represses GATA-1, the major transcriptional regulator of erythropoiesis. We used RNA interference
to ask whether inhibiting PU.1 synthesis was sufficient to cause MEL cells to lose their malignant properties. We report here
that transfection of MEL cells with a PU.1-specific short interfering RNA oligonucleotide causes the cells to resume erythroid
differentiation, accumulate hemoglobin, and undergo terminal growth arrest. RNA interference directed at specific, aberrantly
expressed transcription factors may hold promise for the development of potent antitumor therapies in other hematologic malignancies.
(Mol Cancer Res 2007;5(10):1053–62)
Some tumor cells can be stimulated to differentiate and undergo terminal cell division and loss of tumorigenicity. The in vitro differentiation of murine erythroleukemia (MEL) cells is a dramatic ...example of tumor-cell reprogramming. We found that reentry of MEL cells into terminal differentiation is accompanied by an early transient decline in the activity of cyclin-dependent kinase (CDK) 2, followed by a decline of CDK6. Later, as cells undergo terminal arrest, CDK2 and CDK4 activities decline. By analyzing stable MEL-cell transfectants containing vectors directing inducible expression of specific CDK inhibitors, we show that only inhibitors that block the combination of CDK2 and CDK6 trigger differentiation. Inhibiting CDK2 and CDK4 does not cause differentiation. Importantly, we also show that reprogramming through inhibition of CDKs is restricted to G1phase of the cell cycle. The results imply that abrogation of normal cell-cycle controls in tumor cells contributes to their inability to differentiate fully and that restoration of such controls in G1can lead to resumption of differentiation and terminal cell division. The results also indicate that CDK4 and CDK6 are functionally distinct and support our hypothesis that the two CDKs regulate cell division at different stages of erythroid maturation.
Hematopoietic transcription factors GATA-1 and PU.1 bind each other on DNA to block transcriptional programs of undesired
lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells ...that coexpress GATA-1 and PU.1 are blocked at
the blast stage but respond to molecular removal (downregulation) of PU.1 or addition (upregulation) of GATA-1 by inducing
terminal erythroid differentiation. To test whether GATA-1 blocks PU.1 in MEL cells, we have conditionally activated a transgenic
PU.1 protein fused with the estrogen receptor ligand-binding domain (PUER), resulting in activation of a myeloid transcriptional
program. Gene expression arrays identified components of the PU.1-dependent transcriptome negatively regulated by GATA-1 in
MEL cells, including CCAAT/enhancer binding protein α (Cebpa) and core-binding factor, β subunit (Cbfb), which encode two
key hematopoietic transcription factors. Inhibition of GATA-1 by small interfering RNA resulted in derepression of PU.1 target
genes. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Significant derepression of Cebpa and Cbfb is achieved in MEL cells by either activation of PU.1 or knockdown of GATA-1. Furthermore, transcriptional regulation of
these loci by manipulating the levels of PU.1 and GATA-1 involves quantitative increases in a transcriptionally active chromatin
mark: acetylation of histone H3K9. Collectively, we show that either activation of PU.1 or inhibition of GATA-1 efficiently
reverses the transcriptional block imposed by GATA-1 and leads to the activation of a myeloid transcriptional program directed
by PU.1. (Mol Cancer Res 2009;7(10):1693–703)
Pax6 and c‐Maf regulate multiple stages of mammalian lens development. Here, we identified novel distal control regions (DCRs) of the αA‐crystallin gene, a marker of lens fiber cell differentiation ...induced by FGF‐signaling. DCR1 stimulated reporter gene expression in primary lens explants treated with FGF2 linking FGF‐signaling with αA‐crystallin synthesis. A DCR1/αA‐crystallin promoter (including DCR2) coupled with EGFP virtually recapitulated the expression pattern of αA‐crystallin in lens epithelium and fibers. In contrast, the DCR3/αA/EGFP reporter was expressed only in ‘late’ lens fibers. Chromatin immunoprecipitations showed binding of Pax6 to DCR1 and the αA‐crystallin promoter in lens chromatin and demonstrated that high levels of αA‐crystallin expression correlate with increased binding of c‐Maf and CREB to the promoter and of CREB to DCR3, a broad domain of histone H3K9‐hyperacetylation extending from DCR1 to DCR3, and increased abundance of chromatin remodeling enzymes Brg1 and Snf2h at the αA‐crystallin locus. Our data demonstrate a novel mechanism of Pax6, c‐Maf and CREB function, through regulation of chromatin‐remodeling enzymes, and suggest a multistage model for the activation of αA‐crystallin during lens differentiation.
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