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3D genome mapping aims at connecting the physics of chromatin folding to the underlying biological events, and applications of various chromosomal conformation capture (3C) assays continue to discover critical roles of genome folding in regulating nuclear functions. To interrogate the full spectrum of chromatin folding ranging from the level of nucleosomes to full chromosomes in mammals, we developed an enhanced 3C-based method called Micro-C. The protocol employs Micrococcal nuclease (MNase) to fragment the genome, which overcomes the resolution limit of restriction enzyme-based methods, enabling the estimation of contact frequencies between proximal nucleosomes. Such improvements successfully resolve the fine-scale level of chromatin folding, including enhancer-promoter or promoter-promoter interactions, genic and nucleosomal folding, and boost the signal-to-noise ratio in detecting loops and substructures underlying TADs. In this chapter, we will thoroughly discuss the details of the Micro-C protocol and critical parameters to consider for generating high-quality Micro-C maps.

It remains unclear why acute depletion of CTCF (CCCTC-binding factor) and cohesin only marginally affects expression of most genes despite substantially perturbing three-dimensional (3D) genome folding at the level of domains and structural loops. To address this conundrum, we used high-resolution Micro-C and nascent transcript profiling in mouse embryonic stem cells. We find that enhancer–promoter (E–P) interactions are largely insensitive to acute (3-h) depletion of CTCF, cohesin or WAPL. YY1 has been proposed as a structural regulator of E–P loops, but acute YY1 depletion also had minimal effects on E–P loops, transcription and 3D genome folding. Strikingly, live-cell, single-molecule imaging revealed that cohesin depletion reduced transcription factor (TF) binding to chromatin. Thus, although CTCF, cohesin, WAPL or YY1 is not required for the short-term maintenance of most E–P interactions and gene expression, our results suggest that cohesin may facilitate TFs to search for and bind their targets more efficiently.

It remains unclear why acute depletion of CTCF and cohesin only marginally affects expression of most genes despite substantially perturbing 3D genome folding at the level of domains and structural loops. To address this conundrum, we used high-resolution Micro-C and nascent transcript profiling to find that enhancer-promoter (E-P) interactions are largely insensitive to acute (3-hour) depletion of CTCF, cohesin, and WAPL. YY1 has been proposed to be a structural regulator of E-P loops, but acute YY1 depletion also had minimal effects on E-P loops, transcription, and 3D genome folding. Strikingly, live-cell single-molecule imaging revealed that cohesin depletion reduced transcription factor binding to chromatin. Thus, although neither CTCF, cohesin, WAPL, nor YY1 are required for the short-term maintenance of most E-P interactions and gene expression, we propose that cohesin may serve as a “transcription factor binding platform” that facilitates transcription factor binding to chromatin.

© 2020 Elsevier Inc. Whereas folding of genomes at the large scale of epigenomic compartments and topologically associating domains (TADs) is now relatively well understood, how chromatin is folded at finer scales remains largely unexplored in mammals. Here, we overcome some limitations of conventional 3C-based methods by using high-resolution Micro-C to probe links between 3D genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregates TAD regions into various finer-scale structures with distinct regulatory features including stripes, dots, and domains linking promoters-to-promoters (P-P) or enhancers-to-promoters (E-P) and bundle contacts between Polycomb regions. E-P stripes extending from the edge of domains predominantly link co-expressed loci, often in the absence of CTCF and cohesin occupancy. Acute inhibition of transcription disrupts these gene-related folding features without altering higher-order chromatin structures. Our study uncovers previously obscured finer-scale genome organization, establishing functional links between chromatin folding and gene regulation.

Author(s): Awasthi, Shevya; Das, Doyel; Hardin, Cassidy; Lee, Rosa; Russo, Melanie; Slobodyanyuk, Elena

Author(s): Chari, Nikhil; Lee, Rosa; Mijjum, Moe; De Lorimier, Phillip; Trika, Sona; Slobodyanyuk, Elena

NEUROTRANSMITTER IMAGING AND PLANT NANOBIONICS Interview with Professor Markita Landry BY CATRIN BAILEY, ISABEL CRAIG, NIKHIL CHARI, YANA PETRI, ELENA SLOBODYANYUK Dr. Markita Landry is an Assistant Professor of Chem- ical and Biomedical Engineering at the University of California, Berkeley. Professor Landry’s laboratory focuses on understanding and exploiting optical nanomaterials to access information about biological systems. In this interview, we discuss semiconducting single-walled carbon nanotubes (SWNTs) and their applications in the detection of dopamine in the brain and biological cargo delivery to plant systems. Professor Markita Landry [Source: UC Berkeley College of Chemis- ing biophysics tools in engineering space. That's how I ended up here as well. BSJ Chemical and Biomedical Engineering? : What has made you so interested in optical I trained in Physics for my undergraduate BSJ nanomaterials and nano-sensor design? ML degree and Ph.D. The focus of my Ph.D. work was to study molecular interactions. To do so, There is a lot of opportunity in developing our lab developed high spatial and temporal reso- ML nanosensors, especially for molecules that : How did you first get involved in the field of lution instruments, which were well-suited for the systems that we were studying. When I graduated, I felt that these instruments were more broadly appli- cable and wanted to translate their use into nano- technology. For my postdoc, I planned to come back to physics and then apply nanotechnology tools, but biophysics tools ended up being really useful for nanotechnology. That's how I was introduced to Chemical and Biomolecular Engineering: by build- Berkeley Scientific Journal | SPRING 2017 are otherwise very difficult to access information from. For example, when we diagnose something like cancer, we use quantitative methods: typically, a blood screen for biomarkers and then an assay that shows how many cytokines are in the blood. For behavioral disorders like psychosis and depression, we have only very qualitative methods. That's where my interests are: in the more challenging areas to develop sensors for. I'm trying to make diagnosis

THE ROLE OF ADENO-ASSOCIATED VIRUSES Interview with Professor David Schaffer BY CATRIN BAILEY, YANA PETRI, ELENA SLOBODYANYUK, JORDAN WONG, TIANSHU ZHAO, DANIEL YOON Dr. David Schaffer is a Professor of Chemical and Biomolecular Engineering, Bioengineering, and Neu- roscience at the University of California, Berkeley. Professor Schaffer is interested in stem cell bioengi- neering, gene delivery systems, molecular virology, and their applications to biomedical problems. In this interview, we talk about the role of adeno-associated viruses in gene therapy and discuss its molecular basis and directed evolution approaches. Professor David Schaffer [Source: The Schaffer Lab] BSJ in Chemical and Biomolecular Engineering? BSJ in gene therapy? : How did you first get involved in research DS background both on the basic sciences side : Well, I come from a family with a medical and the clinical side. I was always interested in problems related to human health. I like molecules and I like thinking about problems quantitatively. So, if you put that all together - math, molecules, application towards healthcare - at the time, it was Chemical and Biomolecular Engineering. These days I think that this research takes place in both the CBE department as well as the Bioengineering depart- ment and reflecting that I have an appointment in both. Berkeley Scientific Journal | FALL 2016 : What has inspired your interest specifically DS graduate school (that was a number of years : Well, I began to work in gene therapy during ago, I probably shouldn’t tell you as it is going to date me), so I have been working in the field for over 20 years. At that time, there was a lot of excitement: people were talking about sequencing the human genome, the Human Genome Project was getting underway. The genes that cause haemophilia B, haemophilia A, cystic fibrosis, muscular dystrophy, and Huntington’s disease were getting cloned and se- quenced and it brought up the idea that DNA could be used as a medicine to be able to treat diseases. I thought that this would be revolutionary and got really excited because up until that point many of