The Drosophila melanogaster Nipped-B protein facilitates transcriptional activation of the cut and Ultrabithorax genes by remote enhancers. Sequence homologues of Nipped-B, Scc2 of Saccharomyces cerevisiae, and Mis4 of Schizosaccharomyces pombe are required for sister chromatid cohesion during mitosis. The evolutionarily conserved Cohesin protein complex mediates sister chromatid cohesion, and Scc2 and Mis4 are needed for Cohesin to associate with chromosomes. Here, we show that Nipped-B is also required for sister chromatid cohesion but that, opposite to the effect of Nipped-B, the stromalin/Scc3 component of Cohesin inhibits long-range activation of cut. To explain these findings, we propose a model based on the chromatin domain boundary activities of Cohesin in which Nipped-B facilitates cut activation by alleviating Cohesin-mediated blocking of enhancer-promoter communication.The available evidence, derived largely from studies with Saccharomyces cerevisiae, supports the idea that transcriptional activator proteins recruit basal transcription machinery and chromatin-modifying enzymes to the promoter (43). In essence, activator proteins increase the local concentration of the transcriptional machinery near the promoter. Most yeast activators bind less than a kilobase upstream of the promoters they activate, and looping of the chromatin between the activator and the promoter accommodates interactions. Consistent with this model, the yeast activators tested work poorly when positioned more than a few hundred base pairs upstream of the promoter (53).Although it explains much of yeast gene activation, the localconcentration model does not adequately explain long-range activation by metazoan enhancers, which are often tens of kilobases from the promoter. Based on theoretical calculations, two sequences separated by several kilobases in DNA, or in various forms of chromatin, do not have a local concentration relative to each other greater than that of the estimated total concentration of all the promoters in a nucleus (46). In addition to these theoretical considerations, it was found experimentally that site-specific recombination between two sites separated by several kilobases in mammalian cells occurs slowly compared to recombination between two sites separated by a kilobase or so (45). Thus, an enhancer located several kilobases from a promoter is not expected to contact the promoter specifically or efficiently if it is dependent on random diffusion alone. Studies with the human ␤-globin locus using two different techniques, however, indicate that an enhancer in the locus control region located some 40 to 60 kb from the globin genes is in close proximity to a globin gene promoter when that promoter is activated (8,56). This suggests that mechanisms exist to facilitate enhancer-promoter contact over large distances.Studies of Drosophila melanogaster have identified sequences that facilitate particular long-range enhancer-promoter interactions. The large interval separating the iab-7 enhancer and the Abd-B gene ...

Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A) fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate.

The cohesin protein complex is a conserved structural component of chromosomes. Cohesin binds numerous sites along interphase chromosomes and is essential for sister chromatid cohesion and DNA repair. Here, we test the idea that cohesin also regulates gene expression. This idea arose from the finding that the Drosophila Nipped-B protein, a functional homolog of the yeast Scc2 factor that loads cohesin onto chromosomes, facilitates the transcriptional activation of certain genes by enhancers located many kilobases away from their promoters. We find that cohesin binds between a remote wing margin enhancer and the promoter at the cut locus in cultured cells, and that reducing the dosage of the Smc1 cohesin subunit increases cut expression in the developing wing margin. We also find that cut expression is increased by a unique pds5 gene mutation that reduces the binding of cohesin to chromosomes. On the basis of these results, we posit that cohesin inhibits long-range activation of the Drosophila cut gene, and that Nipped-B facilitates activation by regulating cohesin-chromosome binding. Such effects of cohesin on gene expression could be responsible for many of the developmental deficits that occur in Cornelia de Lange syndrome, which is caused by mutations in the human homolog of Nipped-B.

Key Points Dnmt3a-null hematopoietic stem cells (HSCs) cannot sustain long-term hematopoiesis. Cooperating c-Kit mutations drive leukemic transformation of Dnmt3a-null HSCs.

SUMMARY We developed Split DamID (SpDamID), a protein complementation version of DamID, to mark genomic DNA bound in vivo by interacting or juxtapositioned transcription factors. Inactive halves of DAM (DNA Adenine Methyltransferase) were fused to protein pairs to be queried Interaction or proximity enabled DAM reconstitution and methylation of adenine in GATC. Inducible SpDamID was used to analyze Notch-mediated transcriptional activation. We demonstrate that Notch complexes label RBP sites broadly across the genome, and show that a subset of these complexes that recruit MAML and p300 undergo changes in chromatin accessibility in response to Notch signaling. SpDamID differentiates between monomeric and dimeric binding thereby allowing for identification of half-site motifs used by Notch dimers. Motif enrichment of Notch enhancers coupled with SpDamID reveals co-targeting of regulatory sequences by Notch and Runx1. SpDamID represents a sensitive and powerful tool that enables dynamic analysis of combinatorial protein-DNA transactions at a genome-wide level.