LADL: Light-activated dynamic looping for endogenous gene expression control

Abstract

Mammalian genomes are folded into tens of thousands of long-range looping interactions1,2. The cause and effect relationship between looping and genome function is poorly understood, and the extent to which chromatin loops are dynamic on short time scales remains a fundamental unanswered question. Currently available strategies for loop engineering involve synthetic transcription factors tethered to dCas93,4 or zinc fingers5,6, which are constitutively expressed5,6 or induced on long time scales by the presence of a small molecule3. Here we report a new class of 3-D optoepigenetic tools for the directed rearrangement of 3-D chromatin looping on short time scales using blue light. We create synthetic architectural proteins by fusing the CIBN protein subunit from Arabidopsis thaliana with enzymatically dead Cas9 (dCas9). We target our light-activated dynamic looping system (LADL) to two genomic anchors with CRISPR guide RNAs and engineer their spatial co-localization via light-induced heterodimerization of the cryptochrome 2 (CRY2) protein with dCas9-CIBN. We apply LADL to redirect a stretch enhancer (SE) away from its endogenous Klf4 target gene and to the Zfp462 promoter. Looping changes occur as early as four hours after light induction. Using single molecule RNA FISH, we observe a LADL-induced increase in the total nascent Zfp462 transcripts and the number of Zfp462 alleles expressing simultaneously per cell. Moreover, LADL also increased synchronous Sox2 expression after reinforcement of a known Sox2-SE looping interaction. LADL facilitates loop synchronization across a large population of cells without exogenous chemical cofactors and can enable future efforts to engineer reversible and oscillatory looping on short time scales.

Dynamic simulations of transcriptional control during cell reprogramming reveal spatial chromatin caging

“…The model also proposes that promoter-enhancer communication 45 occurs via direct interactions between the distant enhancer and its cognate target gene as previously observed in many studies 33,37,43,44,[46][47][48][49][50][51][52][53][54] . In summary, our hit-and-stick model links the promoter-enhancer direct contact to the formation of local structural domains or "cages".…”

In silico discovery of repetitive elements as key sequence determinants of 3D genome folding

“…Large structural variants are also rare at domain boundaries in healthy humans but not in patients with autism or developmental delay [11]. To understand the mechanisms underlying these associations, experimental studies have engineered chromatin contact in cells and mice with synthetic tethering [12] and CRISPR systems [13][14][15] and measured their effects on genome folding and expression of genes such as Hbb and Vcan. Findings in these individual loci may not apply genomewide and could overlook mechanisms without known precedent.…”