Christiane Wirbelauer

DNA-binding factors shape the mouse methylome at distal regulatory regions

Methylation of cytosines is an essential epigenetic modification in mammalian genomes, yet the rules that govern methylation patterns remain largely elusive. To gain insights into this process, we generated base-pair-resolution mouse methylomes in stem cells and neuronal progenitors. Advanced quantitative analysis identified low-methylated regions (LMRs) with an average methylation of 30%. These represent CpG-poor distal regulatory regions as evidenced by location, DNase I hypersensitivity, presence of enhancer chromatin marks and enhancer activity in reporter assays. LMRs are occupied by DNA-binding factors and their binding is necessary and sufficient to create LMRs. A comparison of neuronal and stem-cell methylomes confirms this dependency, as cell-type-specific LMRs are occupied by cell-type-specific transcription factors. This study provides methylome references for the mouse and shows that DNA-binding factors locally influence DNA methylation, enabling the identification of active regulatory regions.

p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells.

The F-box protein p45SKP2 is the substrate-targeting subunit of the ubiquitin–protein ligase SCFSKP2 and is frequently overexpressed in transformed cells. Here we report that expression of p45SKP2 in untransformed fibroblasts activates DNA synthesis in cells that would otherwise growth-arrest. Expression of p45SKP2 in quiescent fibroblasts promotes p27Kip1 degradation, allows the generation of cyclin-A-dependent kinase activity and induces S phase. Coexpression of a degradation-resistant p27Kip1 mutant suppresses p45SKP2-induced cyclin-A-kinase activation and S-phase entry. We propose that p45SKP2 is important in the progression from quiescence to S phase and that the ability of p45SKP2 to promote p27Kip1 degradation is a key aspect of its S-phase-inducing function. In transformed cells, p45SKP2 may contribute to deregulated initiation of DNA replication by interfering with p27Kip1 function.

Interaction between ubiquitin–protein ligase SCF SKP2 and E2F-1 underlies the regulation of E2F-1 degradation

The transcription factor E2F-1 is important in the control of cell proliferation. Its activity must be tightly regulated in a cell-cycle-dependent manner to enable programs of gene expression to be coupled closely with cell-cycle position. Here we show that, following its accumulation in the late G1 phase of the cell cycle, E2F-1 is rapidly degraded in S/G2 phase. This event is linked to a specific interaction of E2F-1 with the F-box-containing protein p45SKP2, which is the cell-cycle-regulated component of the ubiquitin–protein ligase SCFSKP2 that recognizes substrates for this ligase. Disruption of the interaction between E2F-1 and p45SKP2 results in a reduction in ubiquitination of E2F-1 and the stabilization and accumulation of transcriptionally active E2F-1 protein. These results indicate that an SCFSKP2-dependent ubiquitination pathway may be involved in the downregulation of E2F-1 activity in the S/G2 phase of the cell cycle, and suggest a link between SCFSKP2 and cell-cycle-dependent gene control.

Identification of genetic elements that autonomously determine DNA methylation states

Cytosine methylation is a repressive, epigenetically propagated DNA modification. Although patterns of DNA methylation seem tightly regulated in mammals, it is unclear how these are specified and to what extent this process entails genetic or epigenetic regulation. To dissect the role of the underlying DNA sequence, we sequentially inserted over 50 different DNA elements into the same genomic locus in mouse stem cells. Promoter sequences of approximately 1,000 bp autonomously recapitulated correct DNA methylation in pluripotent cells. Moreover, they supported proper de novo methylation during differentiation. Truncation analysis revealed that this regulatory potential is contained within small methylation-determining regions (MDRs). MDRs can mediate both hypomethylation and de novo methylation in cis, and their activity depends on developmental state, motifs for DNA-binding factors and a critical CpG density. These results demonstrate that proximal sequence elements are both necessary and sufficient for regulating DNA methylation and reveal basic constraints of this regulation.

Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation.

DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined genomic binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG-dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.

Association of human CUL‐1 and ubiquitin‐conjugating enzyme CDC34 with the F‐box protein p45SKP2: evidence for evolutionary conservation in the subunit composition of the CDC34–SCF pathway

In normal and transformed cells, the F‐box protein p45SKP2 is required for S phase and forms stable complexes with p19SKP1 and cyclin A–cyclin‐dependent kinase (CDK)2. Here we identify human CUL‐1, a member of the cullin family, and the ubiquitin‐conjugating enzyme CDC34 as additional partners of p45SKP2 in vivo. CUL‐1 also associates with cyclin A and p19SKP1 in vivo and, with p45SKP2, they assemble into a large multiprotein complex. In Saccharomyces cerevisiae, a complex of similar molecular composition (an F‐box protein, a member of the cullin family and a homolog of p19SKP1) forms a functional E3 ubiquitin protein ligase complex, designated SCFCDC4, that facilitates ubiquitination of a CDK inhibitor by CDC34. The data presented here imply that the p45SKP2–CUL‐1–p19SKP1 complex may be a human representative of an SCF‐type E3 ubiquitin protein ligase. We propose that all eukaryotic cells may use a common ubiquitin conjugation apparatus to promote S phase. Finally, we show that multiprotein complex formation involving p45SKP2–CUL‐1 and p19SKP1 is governed, in part, by periodic, S phase‐specific accumulation of the p45SKP2 subunit and by the p45SKP2‐bound cyclin A–CDK2. The dependency of p45SKP2–p19SKP1 complex formation on cyclin A–CDK2 may ensure tight coordination of the activities of the cell cycle clock with those of a potential ubiquitin conjugation pathway.

The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts

The ubiquitin protein ligase SCFSkp2 is composed of Skp1, Cul1, Roc1/Rbx1 and the F‐box protein Skp2, the substrate‐recognition subunit. Levels of Skp2 decrease as cells exit the cell cycle and increase as cells re‐enter the cycle. Ectopic expression of Skp2 in quiescent fibroblasts causes mitogen‐independent S‐phase entry. Hence, mechanisms must exist for limiting Skp2 protein expression during the G0/G1 phases. Here we show that Skp2 is degraded by the proteasome in G0/G1 and is stabilized when cells re‐enter the cell cycle. Rapid degradation of Skp2 in quiescent cells depends on Skp2 sequences that contribute to Cul1 binding and interference with endogenous Cul1 function in serum‐deprived cells induces Skp2 expression. Furthermore, recombinant Cul1–Roc1/Rbx1–Skp1 complexes can catalyse Skp2 ubiquitylation in vitro. These results suggest that degradation of Skp2 in G0/G1 is mediated, at least in part, by an autocatalytic mechanism involving a Skp2‐bound Cul1‐based core ubiquitin ligase and imply a role for this mechanism in the suppression of SCFSkp2 ubiquitin protein ligase function during the G0/G1 phases of the cell cycle.

The HRPT2 Tumor Suppressor Gene Product Parafibromin Associates with Human PAF1 and RNA Polymerase II

ABSTRACT Inactivation of the HRPT2 tumor suppressor gene is associated with the pathogenesis of the hereditary hyperparathyroidism-jaw tumor syndrome and malignancy in sporadic parathyroid tumors. The cellular function of the HPRT2 gene product, parafibromin, has not been defined yet. Here we show that parafibromin physically interacts with human orthologs of yeast Paf1 complex components, including PAF1, LEO1, and CTR9, that are involved in transcription elongation and 3′ end processing. It also associates with modified forms of the large subunit of RNA polymerase II, in particular those phosphorylated on serine 5 or 2 within the carboxy-terminal domain, that are important for the coordinate recruitment of transcription elongation and RNA processing machineries during the transcription cycle. These interactions depend on a C-terminal domain of parafibromin, which is deleted in ca. 80% of clinically relevant mutations. Finally, RNAi-induced downregulation of parafibromin promotes entry into S phase, implying a role for parafibromin as an inhibitor of cell cycle progression. Taken together, these findings link the tumor suppressor parafibromin to the transcription elongation and RNA processing pathway as a PAF1 complex- and RNA polymerase II-bound protein. Dysfunction of this pathway may be a general phenomenon in the majority of cases of hereditary parathyroid cancer.

Localized H3K36 methylation states define histone H4K16 acetylation during transcriptional elongation in Drosophila.

Post‐translational modifications of histones are involved in transcript initiation and elongation. Methylation of lysine 36 of histone H3 (H3K36me) resides promoter distal at transcribed regions in Saccharomyces cerevisiae and is thought to prevent spurious initiation through recruitment of histone‐deacetylase activity. Here, we report surprising complexity in distribution, regulation and readout of H3K36me in Drosophila involving two histone methyltransferases (HMTases). Dimethylation of H3K36 peaks adjacent to promoters and requires dMes‐4, whereas trimethylation accumulates toward the 3′ end of genes and relies on dHypb. Reduction of H3K36me3 is lethal in Drosophila larvae and leads to elevated levels of acetylation, specifically at lysine 16 of histone H4 (H4K16ac). In contrast, reduction of both di‐ and trimethylation decreases lysine 16 acetylation. Thus di‐ and trimethylation of H3K36 have opposite effects on H4K16 acetylation, which we propose enable dynamic changes in chromatin compaction during transcript elongation.

A chromatin-modifying function of JNK during stem cell differentiation

Signaling mediates cellular responses to extracellular stimuli. The c-Jun NH2-terminal kinase (JNK) pathway exemplifies one subgroup of the mitogen-activated protein (MAP) kinases, which, besides having established functions in stress response, also contribute to development by an unknown mechanism. We show by genome-wide location analysis that JNK binds to a large set of active promoters during the differentiation of stem cells into neurons. JNK-bound promoters are enriched with binding motifs for the transcription factor NF-Y but not for AP-1. NF-Y occupies these predicted sites, and overexpression of dominant-negative NF-YA reduces the JNK presence on chromatin. We find that histone H3 Ser10 (H3S10) is a substrate for JNK, and JNK-bound promoters are enriched for H3S10 phosphorylation. Inhibition of JNK signaling in post-mitotic neurons reduces phosphorylation at H3S10 and the expression of target genes. These results establish MAP kinase binding and function on chromatin at a novel class of target genes during stem cell differentiation.