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The transcription factor Miz1 forms repressive DNA-binding complexes with the Myc, Gfi-1 and Bcl-6 oncoproteins. Known target genes of these complexes encode the cyclin-dependent kinase inhibitors (CKIs) cdkn2b (p15\(^{Ink4}\)), cdkn1a (p21\(^{Cip1}\)), and cdkn1c (p57\(^{Kip2}\)). Whether Miz1-mediated repression is important for control of cell proliferation in vivo and for tumor formation is unknown. Here we show that deletion of the Miz1 POZ domain, which is critical for Miz1 function, restrains the development of skin tumors in a model of chemically-induced, Ras-dependent tumorigenesis. While the stem cell compartment appears unaffected, interfollicular keratinocytes lacking functional Miz1 exhibit a reduced proliferation and an accelerated differentiation of the epidermis in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Tumorigenesis, proliferation and normal differentiation are restored in animals lacking cdkn1a, but not in those lacking cdkn2b. Our data demonstrate that Miz1-mediated attenuation of cell cycle arrest pathways via repression of cdkn1a has a critical role during tumorigenesis in the skin.
Background: Because most human stroke victims are elderly, studies of experimental stroke in the aged rather than the young rat model may be optimal for identifying clinically relevant cellular responses, as well for pinpointing beneficial interventions.
Methodology/Principal Findings: We employed the Affymetrix platform to analyze the whole-gene transcriptome following temporary ligation of the middle cerebral artery in aged and young rats. The correspondence, heat map, and dendrogram analyses independently suggest a differential, age-group-specific behaviour of major gene clusters after stroke. Overall, the pattern of gene expression strongly suggests that the response of the aged rat brain is qualitatively rather than quantitatively different from the young, i.e. the total number of regulated genes is comparable in the two age groups, but the aged rats had great difficulty in mounting a timely response to stroke. Our study indicates that four genes related to neuropathic syndrome, stress, anxiety disorders and depression (Acvr1c, Cort, Htr2b and Pnoc) may have impaired response to stroke in aged rats. New therapeutic options in aged rats may also include Calcrl, Cyp11b1, Prcp, Cebpa, Cfd, Gpnmb, Fcgr2b, Fcgr3a, Tnfrsf26, Adam 17 and Mmp14. An unexpected target is the enzyme 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 in aged rats, a key enzyme in the cholesterol synthesis pathway. Post-stroke axonal growth was compromised in both age groups.
Conclusion/Significance: We suggest that a multi-stage, multimodal treatment in aged animals may be more likely to produce positive results. Such a therapeutic approach should be focused on tissue restoration but should also address other aspects of patient post-stroke therapy such as neuropathic syndrome, stress, anxiety disorders, depression, neurotransmission and blood pressure.
Hey1, Hey2 and HeyL are downstream effectors of the Notch signalling pathway. Hey genes play decisive roles during embryonic development for example in cardiovascular development. However, the precise transcriptional programmes and genes, which are affected by each single Hey gene, are still poorly understood. One drawback for the analysis of Hey1, Hey2 or HeyL single gene function is that these genes are co-expressed in many tissues and share a high degree of functional redundancy. Thus, it was necessary to establish a system, which is either devoid of Hey expression, or just comprises one single Hey gene family member. For this, Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- as well as Hey-triple- knock out (KO)-ES cells (embryonic stem cells) were generated in this work, because ES cells and their differentiation as EBs (embryoid bodies) represent a valuable tool for the in vitro analysis of embryonic developmental processes. After the establishment of Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- and Hey-triple- KO-ES cells, it could be seen by ALP staining and pluripotency marker expression that loss of Hey expression did not affect ES cell pluripotency features. Thus, these ES cells represent bona fide ES cells and could be further used for the differentiation as EBs. Here, differences in gene expression between Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- and Hey-triple- KO-ES cells (after the loss of Hey1) could be observed in realtime-RT-PCR analysis for the endodermal marker AFP as well as for neural and myogenic markers in d10 EBs. However, the establishment of inducible Hey1, Hey2 or HeyL ES cell lines will be essential to confirm these findings and to search for novel Hey target genes. To get further insight into the mode of Hey action, the analysis of Hey interaction partners is necessary. One such binding partner, the Bre protein, has previously been found in a yeast-two-hybrid screen. Bre has been described to be a member of two distinct complexes (i.e. the nuclear BRCA1-A complex with a function in DNA damage response and the cytoplasmic BRISC complex), to directly interact with the TNF-receptor and Fas and to interfere with apoptotic signalling. The Hey-Bre interaction could be further corroborated in this work; yet, it was not possible to narrow down the interaction site of Bre with Hey1. It rather seems that non-overlapping parts of the Bre protein may bind to Hey. This interaction may be direct– pointing to more than one interaction site inside the Bre protein – or via a common binding partner such as the endogenous Bre protein itself. Besides the interaction studies, functional assays were performed for a more detailed characterisation of Hey1 and Bre interaction. Here, it could be shown that Hey1 over-expression did not have any influence on Bre sub-cellular localisation. Interestingly, it could be demonstrated that Bre positively interfered with Hey1 repressive function in luciferase assays at three of four promoters analysed. Moreover, interaction with Bre seems to lead to a stabilisation of Hey1. As Bre has been described to modulate the E3-ligase activity intrinsic to the BRCC complex it was analysed whether Bre over-expression results in an ubiquitination of Hey1. Yet, this could not be observed in the present work. Furthermore, an interaction of Bre with ubiquitinated proteins could not be demonstrated in an ubiquitin binding assay. To obtain a better insight into Bre function, Bre LacZ gene trap-ES cells and animals were generated. However, realtime-RT-analyses revealed that these cells and mice did not show a loss of Bre expression on mRNA level indicating that insertion mutagenesis did not occur as expected. However, embryos derived from these mice could nevertheless be used for the detection of tissues with Bre expression by β-galactosidase staining. Bre deficiency on mRNA levels was only achieved after the deletion of the floxed exon 3 resulting in the generation of Bre del-mice. Bre del-mice were fertile and without any obvious phenotype and they were used for the generation of Bre del- and wt-MEFs (murine embryonic fibroblasts). Characterisation of these cells showed that proliferation was not affected after loss of Bre (neither under normal nor under stress conditions). However, loss of Bre notably resulted in a reduction in the BRCA1 DNA damage response, in a slightly increased sensitivity towards apoptosis induction by FasL treatment and in an increase in the K63-poly-ubiquitin content in Bre del-cytoplasmic fractions, probably linked to a change in the BRISC de-ubiquitinase activity. Even though these results have the same tendencies as observed in former studies, the effects in the present work are less striking. Further studies as well as intercrossing of Bre del- to Hey KO-animals will be necessary to further understand the functional relevance of Hey and Bre interaction.