Michael Brandeis

The ontogeny of allele-specific methylation associated with imprinted genes in the mouse.

We have investigated the DNA methylation patterns in genomically imprinted genes of the mouse. Both Igf2 and H19 are associated with clear‐cut regions of allele‐specific paternal modification in late embryonic and adult tissues. By using a sensitive PCR assay, it was possible to follow the methylation state of individual HpaII sites in these genes through gametogenesis and embryogenesis. Most of these CpG moieties are not differentially modified in the mature gametes and also become totally demethylated in the early embryo in a manner similar to non‐imprinted endogenous genes. Thus, the overall allele‐specific methylation pattern at these sites must be established later during embryogenesis after the blastula stage. In contrast, sites in an Igf2r gene intron and one CpG residue in the Igf2 upstream region have allele‐specific modification patterns which are established either in the gametes or shortly after fertilization and are preserved throughout pre‐implantation embryogenesis. These studies suggest that only a few DNA modifications at selective positions in imprinted genes may be candidates for playing a role in the maintenance of parental identity during development.

The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase.

We have studied how the cell cycle‐specific oscillations of mitotic B‐type cyclins are generated in mouse fibroblasts. A reporter enzyme comprising the N‐terminus of a B‐type cyclin fused to bacterial chloramphenicol acetyl transferase (CAT) was degraded at the end of mitosis like endogenous cyclins. Point mutations in the destruction box of this construct completely abolished its mitotic instability. When the destructible reporter was driven by the cyclin B2 promoter, CAT activity mimicked the oscillations in the level of the endogenous cyclin B2. These oscillations were largely conserved when the reporter was transcribed constitutively from the SV40 promoter. Pulse‐chase experiments or addition of the proteasome inhibitors lactacystin and ALLN showed that cyclin synthesis continued after the end of mitosis. The destruction box‐specific degradation of cyclins normally ceases at the onset of S phase, and is active in fibroblasts arrested in G0 and in differentiated C2 myoblasts. We were able to reproduce this proteolysis in vitro in extracts of synchronized cells. Extracts of G1 cells degraded cyclin B1 whereas p27Kip1 was stable, in contrast, cyclin B1 remained stable and p27Kip1 was degraded in extracts of S phase cells.

Securin degradation is mediated by fzy and fzr, and is required for complete chromatid separation but not for cytokinesis.

We have studied the ubiquitination and degradation patterns of the human securin/PTTG protein. We show that, in contrast to budding yeast pds1, securin degradation is catalyzed by both fzy (fizzy/cdc20) and fzr (fizzy‐related/cdh1/hct1). Both fzy and fzr also induce the APC/C to ubiquitinate securin in vitro. Securin degradation is mediated by an RXXL destruction box and a KEN box, and is inhibited only when both sequences are mutated. Interestingly, the non‐degradable securin mutant is also partially ubiquitinated by fzy and fzr in vitro. Expressing the non‐degradable securin mutant in cells frequently resulted in incomplete chromatid separation and gave rise to daughter cells connected by a thin chromatin fiber, presumably of chromosomes that failed to split completely. Strikingly, the mutant securin did not prevent the majority of sister chromatids from separating completely, nor did it prevent mitotic cyclin degradation and cytokinesis. This phenotype, reminiscent of the fission yeast cut (cells untimely torn) phenotype, is reported here for the first time in mammals.

Genome-wide transcriptional analysis of the human cell cycle identifies genes differentially regulated in normal and cancer cells

Characterization of the transcriptional regulatory network of the normal cell cycle is essential for understanding the perturbations that lead to cancer. However, the complete set of cycling genes in primary cells has not yet been identified. Here, we report the results of genome-wide expression profiling experiments on synchronized primary human foreskin fibroblasts across the cell cycle. Using a combined experimental and computational approach to deconvolve measured expression values into “single-cell” expression profiles, we were able to overcome the limitations inherent in synchronizing nontransformed mammalian cells. This allowed us to identify 480 periodically expressed genes in primary human foreskin fibroblasts. Analysis of the reconstructed primary cell profiles and comparison with published expression datasets from synchronized transformed cells reveals a large number of genes that cycle exclusively in primary cells. This conclusion was supported by both bioinformatic analysis and experiments performed on other cell types. We suggest that this approach will help pinpoint genetic elements contributing to normal cell growth and cellular transformation.

Indirect inhibition of 26S proteasome activity in a cellular model of Huntington’s disease

Rather than directly impairing 26S proteasomes, misfolded huntingtin may disrupt cellular proteostasis and lead to competition for limited 26S proteasome capacity.

Timing of APC/C substrate degradation is determined by fzy/fzr specificity of destruction boxes

The anaphase promoting complex/cyclosome (APC/C), activated by fzy and fzr, degrades cell cycle proteins that carry RXXL or KEN destruction boxes (d‐boxes). APC/C substrates regulate sequential events and must be degraded in the correct order during mitosis and G1. We studied how d‐boxes determine APC/Cfzy/APC/Cfzr specificity and degradation timing. Cyclin B1 has an RXXL box and is degraded by both APC/Cfzy and APC/Cfzr; fzy has a KEN box and is degraded by APC/Cfzr only. We characterized the degradation of substrates with swapped d‐boxes. Cyclin B1 with KEN was degraded by APC/Cfzr only. Fzy with RXXL could be degraded by APC/Cfzy and APC/Cfzr. Interestingly, APC/Cfzy‐ but not APC/Cfzr‐specific degradation is highly dependent on the location of RXXL. We studied degradation of tagged substrates in real time and observed that APC/Cfzr is activated in early G1. These observations demonstrate how d‐box specificities of APC/Cfzy and APC/Cfzr, and the successive activation of APC/C by fzy and fzr, establish the temporal degradation pattern. Our observations can explain further why some endogenous RXXL substrates are degraded by APC/Cfzy, while others are restricted to APC/Cfzr.

A CDE/CHR tandem element regulates cell cycle‐dependent repression of cyclin B2 transcription

Cyclin B is an important regulator of progression through the cell division cycle. The oscillating appearance of cyclin B1 and B2 proteins during the cell cycle is in part due to fluctuating mRNA levels. We had identified earlier a tandem promoter element named cell cycle‐dependent element (CDE) and cell cycle genes homology region (CHR) which regulates cell cycle‐dependent transcription of cdc25C, cyclin A and cdc2. Here we describe that cyclin B2 transcription is repressed through a novel CDE/CHR element in resting and G1 cells. By relief of this repression in S and G2 oscillating expression of cyclin B2 mRNA is achieved during the cell cycle.

Ubiquitin conjugation triggers misfolded protein sequestration into quality control foci when Hsp70 chaperone levels are limiting

Ubiquitylation of partially misfolded proteins by the yeast Doa10 E3 ligase requires the Hsp40 cochaperone Sis1, whereas the Hsp70 chaperones Ssa1 and Ssa2 are dispensable. Elimination of the Hsp70 chaperones prevents proteasomal degradation, resulting in ubiquitin-dependent sequestration of the misfolded proteins in Hsp42-positive foci.

The DNA Damage Response Mediator MDC1 Directly Interacts with the Anaphase-promoting Complex/Cyclosome

MDC1 (NFBD1), a mediator of the cellular response to DNA damage, plays an important role in checkpoint activation and DNA repair. Here we identified a cross-talk between the DNA damage response and cell cycle regulation. We discovered that MDC1 binds the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that controls the cell cycle. The interaction is direct and is mediated by the tandem BRCA1 C-terminal domains of MDC1 and the C terminus of the Cdc27 (APC3) subunit of the APC/C. It requires the phosphorylation of Cdc27 and is enhanced after induction of DNA damage. We show that the tandem BRCA1 C-terminal domains of MDC1, known to directly bind the phosphorylated form of histone H2AX (γ-H2AX), also bind the APC/C by the same mechanism, as phosphopeptides that correspond to the C termini of γ-H2AX and Cdc27 competed with each other for the binding to MDC1. Our results reveal a link between the cellular response to DNA damage and cell cycle regulation, suggesting that MDC1, known to have a role in checkpoint regulation, executes part of this role by binding the APC/C.

The Stil protein regulates centrosome integrity and mitosis through suppression of Chfr

Stil (Sil, SCL/TAL1 interrupting locus) is a cytosolic and centrosomal protein expressed in proliferating cells that is required for mouse and zebrafish neural development and is mutated in familial microcephaly. Recently the Drosophila melanogaster ortholog of Stil was found to be important for centriole duplication. Consistent with this finding, we report here that mouse embryonic fibroblasts lacking Stil are characterized by slow growth, low mitotic index and absence of clear centrosomes. We hypothesized that Stil regulates mitosis through the tumor suppressor Chfr, an E3 ligase that blocks mitotic entry in response to mitotic stress. Mouse fibroblasts lacking Stil by genomic or RNA interference approaches, as well as E9.5 Stil−/− embryos, express high levels of the Chfr protein and reduced levels of the Chfr substrate Plk1. Exogenous expression of Stil, knockdown of Chfr or overexpression of Plk1 reverse the abnormal mitotic phenotypes of fibroblasts lacking Stil. We further demonstrate that Stil increases Chfr auto-ubiquitination and reduces its protein stability. Thus, Stil is required for centrosome organization, entry into mitosis and cell proliferation, and these functions are at least partially mediated by Chfr and its targets. This is the first identification of a negative regulator of the Chfr mitotic checkpoint.