Argyris Papantonis, Professor for Translational Epigenetics
University Medical Center Goettingen

Homotypic chromatin interactions and loop extrusion are thought to be the two main drivers of mammalian chromosome folding. Over the last years we have been testing the role of RNA polymerase II (RNAPII) across different scales of interphase chromatin organization in a cellular system allowing for its rapid, auxin-mediated degradation. We combine Micro-C and computational modeling to characterize subsets of loops differentially gained or lost upon RNAPII depletion. Our findings reconcile the role of RNAPII in transcription with a direct involvement in setting-up regulatory three-dimensional chromatin contacts genome wide, while also revealing an impact on cohesin loop extrusion.

Dr. Sanalkumar Rajendran Subhadra
Scientist, Genentech

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. We evaluated how these oncogenic fusion proteins orchestrate tumor-specific transcriptional programs by reshaping the 3D chromatin connectivity of precursor cells. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-fusion binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, onco-fusion protein depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns matching those observed in its precursor cells. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs.

Dovetail’s Vision for 3D Genomics and New Developments

Dr. Lisa Munding
Director of Research and Development, Cantata Bio

Lisa presents Dovetail’s vision for 3D Genomics and the latest scientific developments.  

 

Mukulika Ray, Postdoctoral Researcher
Brown University

Temperature is one important abiotic regulator of gene expression that substantially affects biological functions. How increasing temperature affects gene transcription has not only helped us understand the process of transcription per se but is vital for managing the effects of global changes in temperature on living organisms, including humans, plants, and animals, for economic purposes.

Heat stress (HS), caused by increasing temperature beyond physiological limits, leads to activation of stress response genes and repression of constitutive gene expression, both essential for cell survival. Heat shock is an excellent model for understanding the mechanisms of transcription. A key question is: How are genes specifically targeted for activation or repression upon heat stress? Although extensive studies have elucidated the mechanisms that drive HS-induced activation of stress response genes, little is known about the mechanisms that repress thousands of constitutive genes. The mechanisms of heat shock-regulated transcription have been first and most thoroughly studied in Drosophila which we used as our model system where the GA-binding TF GAF was shown to be essential for activation of genes upon heat stress. Here, we show that another GA-binding transcription factor (TF) protein CLAMP (Chromatin-linked adaptor for MSL complex proteins) that can compete with GAF for its binding sites is essential for repression of constitutive genes upon heat stress. We also identified CLAMP-associated 3D loop anchors using Hi-ChIP both before and after heat shock. Furthermore, CLAMP-bound loop anchors that overlap with genes that are normally actively transcribed and and then become repressed after heat shock are characterized by a different set of chromatin marks and interacting TFs than, those which are normally paused and become activated after HS. Overall, we propose that the function of different GA-binding TFs in mediating 3D interactions distinguish genes that are activated from those that are repressed upon heat stress.

Dr. Myriam El Khawand
Head of Customer Success, Cantata Bio

Myriam takes you through setting up and running a 3D Genomics experiment.

Dr. Cory Padilla
Product Management, Cantata Bio

Find out how we are making the best-in-class Micro-C assay more accessible with new sample types.

Dr. Dan Filipescu
Ph.D., Assistant Professor, Mt. Sinai

MacroH2A has established tumour suppressive functions in melanoma and other cancers, but an unappreciated role in the tumour microenvironment. Using an autochthonous, immunocompetent mouse model of melanoma, we demonstrate that mice devoid of macroH2A variants exhibit increased tumour burden compared with wild-type counterparts. MacroH2A-deficient tumours accumulate immunosuppressive monocytes and are depleted of functional cytotoxic T cells, characteristics consistent with a compromised anti-tumour response. Single cell and spatial transcriptomics identify increased dedifferentiation along the neural crest lineage of the tumour compartment and increased frequency and activation of cancer-associated fibroblasts following macroH2A loss. Mechanistically, macroH2A-deficient cancer-associated fibroblasts display increased myeloid chemoattractant activity as a consequence of hyperinducible expression of inflammatory genes, which is enforced by increased chromatin looping of their promoters to enhancers that gain H3K27ac. In summary, we reveal a tumour suppressive role for macroH2A variants through the regulation of chromatin architecture in the tumour stroma with potential implications for human melanoma.

Dr. Richard Sallari, CEO, Axiotl
Maharshi Chakraborty, Research Lead, Axiotl
Laura Morgan, Platform Lead, Axiotl

At Axiotl we help researchers find hidden patterns in their data.
Using three vignettes, taken from ongoing research with clients and collaborators, we will motivate the design of the Tinker platform, a collection of tools and services built from the ground up for 3D genomics. 
In each vignette, we’ll show how we’re using the platform to explore, interpret and discover patterns of variation in the 3D architecture of epigenomes. In pediatric cancers, we’re visualizing H3K27ac HiChIP to shed light on the effect of HDAC inhibitors on superenhancers. In colon cancer, we’re analyzing tumor-normal matched HiChIP to uncover the effect of reactivated L1s on oncogenes via the formation of chromatin loops. In opioid use disorder, assembling brain-specific maps of enhancer-TSS connections have allowed us to discover new associated genes by aggregating case-specific losses in enhancer activity in the frontal cortex of individuals who suffered overdoses.
Each research question presents a unique challenge that stems from the 3D nature of the data, and each question illustrates how our suite of command line tools, developed in collaboration with the Gryder lab at CWRU and providing built-in AQuA and Inherent normalization, can be combined to produce the desired output with a few lines of compact code.
Following the vignettes, our team members will provide an overview of each of the tools and show how they are run from the Tinker platform, an integrated system used through the web that streamlines the processing and analysis of HiChIP data, from FASTQs to figure PDFs.

 

Dr. Jonathon Torchia
Bioinformatics, Cantata Bio

Find out what can cause biases in genome analysis and how to successfully call TAD’s, Loops, and A/B Compartments.

Dayna Challis
Ph. D. Candidate, University of Tasmania

Medulloblastoma accounts for around 20 percent of childhood brain cancers. While radiotherapy is frequently used to treat medulloblastoma, many tumours exhibit radioresistance. Despite being used for more than a century, the exact mechanisms underlying acquired radioresistance remain poorly understood, and the epigenetic fingerprint from radiotherapy has not been extensively characterised. Epigenetic mechanisms are of particular interest in medulloblastoma due to its low mutational load. We hypothesised that medulloblastoma cells undergo epigenetic reprogramming during radiotherapy and that these changes drive resistance. In this study, a radiotherapy naïve cell line (ONS-76), derived from a primary tumour, was used to model radioresponse. Firstly, we assessed the impact of a single 2 gray dose of radiotherapy on the transcriptional and proteomic profile of cells using RNA-seq and mass spectrometry, respectively. Changes in 3D chromatin structure were subsequently measured using Micro-C following both single and fractionated exposure to radiotherapy. In summary, RNA-seq revealed 601 significantly upregulated genes and 372 downregulated genes. Mass spectrometry identified 361 proteins with significantly increased abundance and 217 with decreased abundance. Notably, the intersection of these datasets highlighted 48 shared genes. Analysis of Micro-C data is currently underway and will reveal the epigenetic landscape underpinning changes in gene expression. Preliminary data highlights compartment identity switching, generally from B to A compartments, five days after both single and repeated exposure to radiotherapy. TADs containing several candidate genes were also altered following radiotherapy; however, superficial analyses suggest most changes are occurring at the loop level. This study is the first to evaluate chromatin conformation post-radiotherapy in brain cancer cells. Our findings will help uncover the mechanisms of acquired radioresistance, and importantly, the identified candidates may represent new targets for radiosensitisation in medulloblastoma.

Dr. Cory Padilla
Product Management, Cantata Bio

Find out how we are making the best-in-class Micro-C assay more accessible with new lower sample input volumes.