Technologies that enable targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we describe a programmable, CRISPR-Cas9-based acetyltransferase consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. The fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, leading to robust transcriptional activation of target genes from promoters and both proximal and distal enhancers. Gene activation by the targeted acetyltransferase was highly specific across the genome. In contrast to previous dCas9-based activators, the acetyltransferase activates genes from enhancer regions and with an individual guide RNA. We also show that the core p300 domain can be fused to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a robust tool for manipulating gene regulation.

/pdf/epigenome-editing-by-a-crispr-cas9-based-acetyltransferase-nz3mx0pe78.pdf

Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers

Modification of Enhancer Chromatin: What, How, and Why?

Transcriptional Regulation and Its Misregulation in Disease

CRISPR is becoming an indispensable tool in biological research. Once known as the bacterial immune system against invading viruses, the programmable capacity of the Cas9 enzyme is now revolutionizing diverse fields of medical research, biotechnology, and agriculture. CRISPR-Cas9 is no longer just a gene-editing tool; the application areas of catalytically impaired inactive Cas9, including gene regulation, epigenetic editing, chromatin engineering, and imaging, now exceed the gene-editing functionality of WT Cas9. Here, we will present a brief history of gene-editing tools and describe the wide range of CRISPR-based genome-targeting tools. We will conclude with future directions and the broader impact of CRISPR technologies.

/pdf/the-crispr-tool-kit-for-genome-editing-and-beyond-j01oba36f4.pdf

The CRISPR tool kit for genome editing and beyond.

How DNA is organized in three dimensions inside the cell nucleus and how that affects the ways in which cells access, read and interpret genetic information are among the longest standing questions in cell biology. Using newly developed molecular, genomic, and computational approaches based on the chromosome conformation capture technology (such as 3C, 4C, 5C and Hi-C) the spatial organization of genomes is being explored at unprecedented resolution. Interpreting the increasingly large chromatin interaction datasets is now posing novel challenges. Here we describe several types of statistical and computational approaches that have recently been developed to analyze chromatin interaction data.

/pdf/exploring-the-three-dimensional-organization-of-genomes-36n1pxvxrk.pdf

Exploring the three-dimensional organization of genomes: interpreting chromatin interaction data

Reactivation of developmentally silenced globin genes by forced chromatin looping.

Enhancer and promoter interactions-long distance calls.

Many questions remain about how close association of genes and distant enhancers occurs and how this is linked to transcription activation. In erythroid cells, lim domain binding 1 (LDB1) protein is recruited to the β-globin locus via LMO2 and is required for looping of the β-globin locus control region (LCR) to the active β-globin promoter. We show that the LDB1 dimerization domain (DD) is necessary and, when fused to LMO2, sufficient to completely restore LCR-promoter looping and transcription in LDB1-depleted cells. The looping function of the DD is unique and irreplaceable by heterologous DDs. Dissection of the DD revealed distinct functional properties of conserved subdomains. Notably, a conserved helical region (DD4/5) is dispensable for LDB1 dimerization and chromatin looping but essential for transcriptional activation. DD4/5 is required for the recruitment of the coregulators FOG1 and the nucleosome remodeling and deacetylating (NuRD) complex. Lack of DD4/5 alters histone acetylation and RNA polymerase II recruitment and results in failure of the locus to migrate to the nuclear interior, as normally occurs during erythroid maturation. These results uncouple enhancer-promoter looping from nuclear migration and transcription activation and reveal new roles for LDB1 in these processes.

/pdf/role-of-ldb1-in-the-transition-from-chromatin-looping-to-35k53xklee.pdf

Ivan Krivega

Papers

Reactivation of developmentally silenced globin genes by forced chromatin looping.

Enhancer and promoter interactions-long distance calls.

Role of LDB1 in the transition from chromatin looping to transcription activation

Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/γ-globin looping

The LDB1 Complex Co-opts CTCF for Erythroid Lineage-Specific Long-Range Enhancer Interactions