Expression of the proneural gene Neurogenin2 is controlled by several enhancer elements, with the E1 element active in restricted progenitor domains in the embryonic spinal cord and telencephalon that express the homeodomain protein Pax6. We show that Pax6 function is both required and sufficient to activate this enhancer, and we identify one evolutionary conserved sequence in the E1 element with high similarity to a consensus Pax6 binding site. This conserved sequence binds Pax6 protein with low affinity both in vitro and in vivo, and its disruption results in a severe decrease in E1 activity in the spinal cord and in its abolition in the cerebral cortex. The regulation of Neurogenin2 by Pax6 is thus direct. Pax6 is expressed in concentration gradients in both spinal cord and telencephalon. We demonstrate that the E1 element is only activated by high concentrations of Pax6 protein, and that this requirement explains the restriction of E1 enhancer activity to domains of high Pax6 expression levels in the medioventral spinal cord and lateral cortex. By modifying the E1 enhancer sequence, we also show that the spatial pattern of enhancer activity is determined by the affinity of its binding site for Pax6. Together, these data demonstrate that direct transcriptional regulation accounts for the coordination between mechanisms of patterning and neurogenesis. They also provide evidence that Pax6 expression gradients are involved in establishing borders of gene expression domains in different regions of the nervous system.

Leukaemogenesis requires enhanced self-renewal, which is induced by oncogenes. The underlying molecular mechanisms remain incompletely understood. Here, we identified C/D box snoRNAs and rRNA 2'-O-methylation as critical determinants of leukaemic stem cell activity. Leukaemogenesis by AML1-ETO required expression of the groucho-related amino-terminal enhancer of split (AES). AES functioned by inducing snoRNA/RNP formation via interaction with the RNA helicase DDX21. Similarly, global loss of C/D box snoRNAs with concomitant loss of rRNA 2'-O-methylation resulted in decreased leukaemia self-renewal potential. Genomic deletion of either C/D box snoRNA SNORD14D or SNORD35A suppressed clonogenic potential of leukaemia cells in vitro and delayed leukaemogenesis in vivo. We further showed that AML1-ETO9a, MYC and MLL-AF9 all enhanced snoRNA formation. Expression levels of C/D box snoRNAs in AML patients correlated closely with in vivo frequency of leukaemic stem cells. Collectively, these findings indicate that induction of C/D box snoRNA/RNP function constitutes an important pathway in leukaemogenesis.

The transcription factors Pax2 and Pax6 are co-expressed in the entire optic vesicle (OV) prior and concomitant with the establishment of distinct neuroretinal, retinal, pigmented-epithelial and optic-stalk progenitor domains, suggesting redundant functions during retinal determination. Pax2; Pax6 compound mutants display a dose-dependent reduction in the expression of the melanocyte determinant Mitf, accompanied by transdifferentiation of retinal pigmented epithelium (RPE) into neuroretina(NR) in Pax2-/-; Pax6+/- embryos,which strongly resembles the phenotype of Mitf-null mutants. In Pax2-/-; Pax6-/- OVs Mitffails to be expressed and NR markers occupy the area that usually represents the Mitf+ RPE domain. Furthermore, both, Pax2 and Pax6 bind to and activate a MITF RPE-promoter element in vitro,whereas prolonged expression of Pax6 in the Pax2-positive optic stalk leads to ectopic Mitf expression and RPE differentiation in vivo. Together,these results demonstrate that the redundant activities of Pax2 and Pax6 direct the determination of RPE, potentially by directly controlling the expression of RPE determinants.

In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-

Vertebrates express two A-type cyclins; both associate with and activate the CDK2 protein kinase. Cyclin A1 is required in the male germ line, but its molecular functions are incompletely understood. We observed specific induction of cyclin A1 expression and promoter activity after UV and ␥-irradiation which was mediated by p53. cyclin A1 ؊/؊ cells showed increased radiosensitivity. To unravel a potential role of cyclin A1 in DNA repair, we performed a yeast triple hybrid screen and identified the Ku70 DNA repair protein as a binding partner and substrate of the cyclin A1-CDK2 complex. DNA double-strand break (DSB) repair was deficient in cyclin A1؊/؊ cells. Further experiments indicated that A-type cyclins activate DNA DSB repair by mechanisms that depend on CDK2 activity and Ku proteins. Both cyclin A1 and cyclin A2 enhanced DSB repair by homologous recombination, but only cyclin A1 significantly activated nonhomologous end joining. DNA DSB repair was specific for A-type cyclins because cyclin E was ineffective. These findings establish a novel function for cyclin A1 and CDK2 in DNA DSB repair following radiation damage.The cell cycle is regulated by external signals, especially in multicellular organisms, where cell proliferation is central to growth and differentiation (34). Internal signals ensure that cell cycle transitions do not occur until all required molecular events have been completed (20). The internal signals become evident as cell cycle checkpoints upon an insult to the cell (e.g., DNA damage) which arrests the cell cycle while repair occurs (10,14). Both internal and external signals modulate the activity of the cyclin-dependent kinases (CDKs) that catalyze the ordered transitions from one phase of the cell cycle to the next. Cyclin E and cyclin A2 both associate with CDK2, and cyclin A2 is essential for DNA replication and proliferation in somatic cells (8). Embryos with a homozygous deletion of cyclin A2 are not capable of proliferative growth (28). During G 1 and S phases, the kinase activity of CDK2 associates first with cyclin E and later with cyclin A2. The timing of these processes as well as the appropriate concentration of cyclin/CDK2 complexes is crucial for successful DNA replication (8).Cyclin A1 is a second A-type cyclin that binds CDK2. Cyclin A1 is abundantly expressed in the testis and has previously been shown to be essential for entry into the metaphase of meiosis I in the male germ line in mice (38, 44). Cyclin A1 is expressed at low levels in most other tissues, but no phenotype other than male infertility has been reported for mice lacking the cyclin A1 gene (19,42). The expression of cyclin A1 in hematopoietic progenitor cells and in acute myeloid leukemia is best characterized in somatic cells (46). Surprisingly, recent microarray data suggested that cyclin A1 was transcriptionally induced following p53 activation (21).Thus, the physiological role of cyclin A1 in somatic cells remains unknown. This prompted us to analyze the mechanisms of cyclin A1 induction in somatic cel...