In NIH3T3 fibroblasts, the ubiquitous helix‐loop‐helix (HLH) protein E2A (E12/E47) and the myogenic HLH proteins MyoD, MRF4 and myogenin are growth‐inhibitory, while two ubiquitous Id proteins lacking the basic region are not. The dimerization domain mediates inhibition. However, in addition to the HLH region, E2A contains two inhibitory regions over‐lapping with the main transcriptional activation domains. The growth‐suppressive activity of the intact E47 as well as MyoD was counteracted by the Id proteins. When E47 lacking the HLH domain was overexpressed, Id could no longer reverse growth inhibition. By increasing the amount of E47 with an inducible system or neutralizing the endogenous Id with microinjected anti‐Id antibodies, withdrawal from the cell cycle occurred within hours before the G1‐S transition point. The combined results suggest that the Id proteins are required for G1 progression. The antagonism between the E2A and Id proteins further suggests that both are involved in regulatory events prior to or near the restriction point in the G1 phase of the cell cycle.

IdM, IdW, and Id3 (HLH462) dimerize with members of the basic helixloophelix protein family, but due to the absence of the basic region, the r uig heterodimers cannot bind DNA. Therefore Id-type proteins negatively regulate DNA binding of the basic helixloop-helix proteins. Here (FCS) and arrested by growing them in medium containing 0.5% FCS. Total RNA was extracted 5, 15, 30, 60, and 360 min after adding 0.5% FCS. After 3 days, when <3% of the cell population was incorporating 5-bromo-2'-deoxyuridine (BrdUrd; Sigma, final concentration, 100 ,ig/ml), arrested cells were induced by addition of medium containing 20%o FCS. Samples were collected at the indicated times. RNA was prepared and blotted as described (18) 4985The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Drosophila kayak mutant embryos exhibit defects in dorsal closure, a morphogenetic cell sheet movement during embryogenesis. Here we show that kayak encodes D-Fos, the Drosophila homologue of the mammalian proto-oncogene product, c-Fos. D-Fos is shown to act in a similar manner to Drosophila Jun: in the cells of the leading edge it is required for the expression of the TGFbeta-like Decapentaplegic (Dpp) protein, which is believed to control the cell shape changes that take place during dorsal closure. Defects observed in mutant embryos, and adults with reduced Fos expression, are reminiscent of phenotypes caused by 'loss of function' mutations in the Drosophila JNKK homologue, hemipterous. These results indicate that D-Fos is required downstream of the Drosophila JNK signal transduction pathway, consistent with a role in heterodimerization with D-Jun, to activate downstream targets such as dpp.

The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation.In 1963 Jacob, Brenner, and Cuzin (24) proposed the replicon model to explain the control of replication of the bacterial chromosome. In this model, DNA replication starts from a specific origin sequence, the replicator, that is recognized by a positive regulatory protein, the initiator. Since then the model has been validated in numerous prokaryotic and viral systems. The organization of the eukaryotic genome in multiple replication units distributed on several chromosomes has hampered the validation of this model in eukaryotes until the identification of the autonomous replicating sequences (ARS) in Saccharomyces cerevisiae. Initially identified for their ability to support the propagation of plasmid molecules in yeast cells, most of these sequences were successively proven to correspond to chromosomal replicators. ARSs are relatively short sequence elements (100 to 200 bp) that include the start site of replication, also called the origin of bidirectional DNA replication (OBR) (11). They consist of an essential 11-bp ARS consensus sequence (ACS) and of several auxiliary B elements that contribute to initiation activity. The ACS binds the origin recognition complex (ORC), a heteromeric complex of six proteins that assists the formation of a prereplicative complex on the origin. ORC orthologs have been isolated from all the eukaryotic species analyzed so far, including humans (for a review see reference 8). The ARS sequences and the ORC can be viewed as the prototypes of the eukaryotic replicator and initiator.The major obstacle to the validation of the replicon model in metazoan cells was the failure to isolate the functional homologues of the ARS elements...

Hox proteins are transcription factors involved in controlling axial patterning, leukaemias and hereditary malformations. Here, we show that HOXC10 oscillates in abundance during the cell cycle, being targeted for degradation early in mitosis by the ubiquitin-dependent proteasome pathway. Among abdominal-B subfamily members, the mitotic proteolysis of HOXC10 appears unique, since the levels of the paralogous HOXD10 and the related homeoprotein HOXC13 are constant throughout the cell cycle. When two destruction box motifs (D-box) are mutated, HOXC10 is stabilized and cells accumulate in metaphase. HOXC10 appears to be a new prometaphase target of the anaphase-promoting complex (APC), since its degradation coincides with cyclin A destruction and is suppressed by expression of a dominant-negative form of UbcH10, an APC-associated ubiquitin-conjugating enzyme. Moreover, HOXC10 co-immunoprecipitates the APC subunit CDC27, and its in vitro degradation is reduced in APC-depleted extracts or by competition with the APC substrate cyclin A. These data imply that HOXC10 is a homeoprotein with the potential to influence mitotic progression, and might provide a link between developmental regulation and cell cycle control.

In as much as AP-1 is instrumental in regulating genes activated at the onset of osteoblast differentiation, such as the ALP gene, we pose that an interplay of distinct MAPKs targeting AP-1 components may dictate the osteogenic response of hPDL cells to mechanical stimulation.

Drosophila Jun (D‐Jun) is a nuclear component of the receptor tyrosine kinase/Ras signal transduction pathway which triggers photoreceptor differentiation during eye development. Here we show that D‐Jun is a substrate for the ERK‐related Drosophila MAP kinase Rolled, which has previously been shown to be a part of this pathway. A D‐Jun mutant that carries alanines in place of the Rolled phosphorylation sites acts as a dominant suppressor of photoreceptor cell fate if expressed in the eye imaginal disc. In contrast, a mutant in which the phosphorylation sites are replaced by phosphate‐mimetic Asp residues, as well as a VP16‐D‐Jun fusion protein, can promote photoreceptor differentiation. These data implicate Jun phosphorylation in the choice between neuronal and non‐neuronal fate during Drosophila eye development.

The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.