Bart Dequeker, PhD Student
Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Vienna, Austria

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs), which contribute to transcription, recombination and genomic stability. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered. Little is known about whether loop extrusion is impeded by DNA-bound macromolecular machines. We demonstrate that the replicative helicase MCM is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C of one-cell embryos revealed that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, suggesting loop extrusion is impeded before reaching CTCF. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Simulations are consistent with MCMs as abundant, random barriers. We conclude that distinct loop extrusion barriers contribute to shaping 3D genomes.

Dr. Melissa Fullwood
(1) School of Biological Sciences, Nanyang Technological University, Singapore (2) Cancer Science Institute of Singapore, National University of Singapore (3) Institute of Molecular and Cell Biology, A*STAR, Singapore

In the first story, we investigate Acute Myeloid Leukemias, which are blood cancers arising from aberrant differentiation of blood cells from haematopoietic stem cells. To date there is no detailed study of 3D genome organization in acute myeloid leukemia patient samples as compared with normal haematopoietic stem cells, which are the precursors of acute myeloid leukemia. Here we performed Hi-C in 3 samples of CD34+-enriched acute myeloid leukemia bone marrow clinical samples and 3 CD34+-enriched bone marrow samples from healthy individuals. Analysis of the Hi-C heatmaps indicated that all the healthy samples and one of the acute myeloid leukemia bone marrow showed an unusual small TAD-shaped interaction that may be a “Frequently Interacting Region” (FIRE) in the MEIS1 locus, which are regions of unusually high local contact frequency that tend to be depleted near Topologically-Associated Domain (TAD) boundaries and enriched towards the center of TADs. The FIRE boundary at MEIS1 is lost in two acute myeloid leukemia bone marrow samples and is associated with loss of MEIS1 gene expression. Recapitulation of the removal of the FIRE boundary at the MEIS1 by CRISPR in K562 myeloid leukemia cells similarly leads to loss of MEIS1 gene expression. Taken together, our analysis of the 3D genome organization in clinical acute myeloid leukemia samples provides a mechanistic explanation underlying MEIS1 oncogene activation in leukemia. In the second story, we investigate silencers by identifying regions with high levels of H3K27me3 signals, similar to the way that super-enhancers are called from regions with high H3K27ac signals. We show that silencers can also loop to target genes. CRISPR excision of silencers leads to gene upregulation and reduced cell survival. Interestingly, CRISPR excision of two silencer components that loop to each other leads to synergistic gene upregulation and greatly diminished cell survival, along with changes in chromatin loops. EZH2 inhibition leads to alterations in 3D genome organization and MRR-associated genes showed high gene upregulation by EZH2. Taken together, our results indicate that silencers may function synergistically via 3D genome organization to silence genes. This information may be helpful for the design of epigenetic drugs to target cancer by modulating enhancers, silencers and chromatin interactions.

Dr Giacomo Cavalli, PI
Montpellier, Occitanie

The eukaryotic genome folds in 3D in a hierarchy of structures, including nucleosomes, chromatin fibers, loops, chromosomal domains (also called TADs), compartments and chromosome territories that are highly organized in order to allow for stable memory as well as for regulatory plasticity, depending on intrinsic and environmental cues. Our lab has provided evidence suggesting that the formation of TADs and chromatin loops can assist gene regulation, both in Drosophila and in mouse cells. Furthermore, cellular stress, such as replicative or oncogene-induced senescence, can induce a massive nuclear reorganization that can affect gene expression. However, the physical nature of compartments, TADs and loops remain elusive and single-cell studies are critically required to understand it. We characterized chromatin folding in single cells using super-resolution microscopy, revealing structural features inaccessible to cell-population analysis. TADs range from condensed and globular objects to stretched conformations. Favored interactions within TADs are regulated by cohesin and CTCF through distinct mechanisms. Furthermore, super-resolution imaging revealed that TADs are subdivided into discrete nanodomains. In order to access the details of chromatin architecture with the best possible resolution we compared HiC with a recently developed method called microC. We found that microC provides sub-kb resolution of chromatin folding, is superior to Hi-C in the detection of chromatin loops and maintains compartmental interaction to a level comparable to Hi-C. We are combining microC to Cut&Run technology in order to reveal the determinants of chromatin topology at an unprecedented level of detail. Altogether, these results provide a physical basis for the folding of individual chromosomes at the nanoscale. Our progress in these fields will be discussed.

Dr. Yasuhiro Murakawa, Professor

Large-scale genome-wide association studies (GWAS) have yielded an increasing number of disease-associated genomic loci. However, the mechanistic interpretation is still far from complete. Recently, it has become apparent that disease-associated genetic variants are often located within enhancers that act to strongly enhance the expression of their target genes in a cell-type specific fashion. Here we have developed a 5’-end single-cell RNA sequencing approach to comprehensively map functional enhancers from heterogeneous helper T cells. By integrating with GWAS datasets, we identified hundreds of human enhancers associated with autoimmune diseases. Furthermore, to gain important clues to human disease pathways, we have identified target genes of these enhancers by using Micro-C, a method that can analyze chromatin interactions with super-high resolution. In sum, we provide a general framework to investigate molecular mechanisms underlying human diseases.

Dr. Berkley Gryder, Assistant Professor
Case Western Reserve University

Mysteriously, a small number of core regulatory transcription factors (CR TFs) control a large fraction of the epigenome. We find these TFs are self-regulated in a special way: using complex 3D folding of large scale and long-range enhancer elements, forming a sort of “combination lock” that restricts them in developmental time and space. Their 3D miswiring causes derailment of enhancer wiring, causing childhood cancer rhabdomyosarcoma. We will discuss how in cancer, these CR TFs are essential, and new insights into how to drug for therapeutic benefit them are emerging. We developed and apply Absolute Quantification of Chromatin Architecture (AQuA) HiChIP to show otherwise hidden features molecules that perturb the levels of histone acetylation.

Dr. Abhijit Parolia, Research Investigator
University of Michigan Medical School

The switch/sucrose non-fermentable (SWI/SNF) complex plays a crucial role in chromatin remodeling and is recurrently altered in over 20% of human cancers. Here, we developed a proteolysis targeting chimera (PROTAC) degrader of ATPase subunits of the SWI/SNF complex, SMARCA2 and SMARCA4. Intriguingly, we found androgen receptor (AR)/forkhead box A1 (FOXA1)-positive prostate cancer and MYC-driven multiple myeloma cell lines to be exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to benign prostate as well as other cancer cell lines, including those with inactivating SMARCA4 mutations. Mechanistically, SWI/SNF inactivation instantaneously compacts the cis-regulatory elements that are bound and activated by transcription factors that drive cancer proliferation, namely AR, FOXA1, ERG, and MYC. This ensues in chromatin untethering of these oncogenic drivers, chemical decommissioning of their core enhancer circuitry, and attenuation of downstream gene programs. Furthermore, we found SWI/SNF inhibition to disrupt super-enhancer and promoter DNA looping interactions that wire supra-physiologic expression of the AR, FOXA1, and MYC oncogenes, thereby tempering their expression in cancer cells. Monotherapy with the SMARCA2/4 degrader induced potent inhibition of tumor growth in cell line-derived xenograft models of prostate cancer and remarkably synergized with AR antagonists, inducing disease remission in models of castration-resistant prostate cancer. We also found the combinatorial treatment to significantly inhibit the growth of enzalutamide-resistant disease using in vitro as well as patient-derived xenograft models. Notably, no major toxicities were seen in mice upon prolonged treatment with the SMARCA2/4 degrader, including no indications of thrombocytopenia, gastrointestinal goblet cell depletion, or germ cell degeneration. Taken together, these results suggest that impeding enhancer accessibility through SWI/SNF ATPase inactivation represents a novel therapeutic approach in enhancer-addicted human cancers.