Cell lines were not tested for mycoplasma contamination. Commonly misidentified lines (See ICLAC register) No commonly misidentified cell lines were used.

Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Heritability and polygenic predictionIn the EUR sample, the SNP-based heritability (h 2 SNP ) (that is, the proportion of variance in liability attributable to all measured SNPs)

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesisdependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serumstimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein ElA. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation. A commonly employed model system for studying reentry into the proliferative state is serum starvation and subsequent stimulation of fibroblasts in culture. Addition of high concentrations of serum or growth factors to quiescent cells leads to a synchronously growing population in which transient activation and repression of growth-responsive genes can be monitored. Between 15 min and 4 h after serum stimulation, the immediate-early genes are expressed. These genes are transcribed in the presence of protein synthesis inhibitors, indicating that preexisting factors in quiescent cells are sufficient for activation of transcription (45). The early-response gene products include transcription factors such as Fos and EGR2. Several hours later (8 to 12 h after serum stimulation), a wave of gene expression requiring protein synthesis occurs at the transition from G, to DNA synthesis (S) phase. This protein synthesis requirement suggests that factors other than those present in quiescent cells are necessary for activation of transcription. Many late-serum-response gene products, such as DNA polymerase alpha, thymidine kinase, thymidylate synthase, carbamoyl phosphate synthase-aspartate carbamoyltransferasedihydrooro...

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Distinct epigenomic profiles of histone marks have been associated with gene expression, but questions regarding the causal relationship remain. Here we investigated the activity of a broad collection of genomically targeted epigenetic regulators that could write epigenetic marks associated with a repressed chromatin state (G9A, SUV39H1, Krüppel-associated box (KRAB), DNMT3A as well as the first targetable versions of Ezh2 and Friend of GATA-1 (FOG1)). dCas9 fusions produced target gene repression over a range of 0- to 10-fold that varied by locus and cell type. dCpf1 fusions were unable to repress gene expression. The most persistent gene repression required the action of several effector domains; however, KRAB-dCas9 did not contribute to persistence in contrast to previous reports. A ‘direct tethering’ strategy attaching the Ezh2 methyltransferase enzyme to dCas9, as well as a ‘recruitment’ strategy attaching the N-terminal 45 residues of FOG1 to dCas9 to recruit the endogenous nucleosome remodeling and deacetylase complex, were both successful in targeted deposition of H3K27me3. Surprisingly, however, repression was not correlated with deposition of either H3K9me3 or H3K27me3. Our results suggest that so-called repressive histone modifications are not sufficient for gene repression. The easily programmable dCas9 toolkit allowed precise control of epigenetic information and dissection of the relationship between the epigenome and gene regulation.