Chromatin organization and transcription cis-regulation in space and time
Keywords: Chemical and Systems Biology.

Genome encodes all the essential information for an organism. They must be safely packaged to ensure the propagation of the genetic materials, while maintaining their accessibility to molecular machineries for proper expression and replication. The need for storing vast amount of genetic information results in a linear length of mammalian genome orders of magnitude longer than the length-scale of a cell. This mismatch of length-scale requires substantial packing of the genomic DNA to fit into the physical space of the cell. In order to achieve the most efficient packaging, the mammalian genome is hierarchically folded across multiple length-scales. Proper read-out of the genomic information is achieved through the process of transcription cis-regulation at the chromatin level, whereby short stretches of non-coding genetic elements -- termed cis-regulatory elements -- coordinate with each other to toggle the on and off of their cognate genes in response to signaling cues. In mammalian cells, cis-regulatory elements regulating the same gene can be separated by large genomic distances. A long-held conundrum in the field of chromatin biology is how do cis-regulatory elements communicate with each other over large genomic distance allowing precise spatio-temporal transcription regulation. Decades-long studies utilizing genetics, biochemistry and molecular biology have accumulated a large body of knowledge regarding the chromatin signatures and function of cis-regulatory elements. However, the biophysical and biochemical nature of transcription cis-regulation remains largely elusive. In the first part of my thesis, I strived to tackle this problem through directly visualizing cis-regulatory elements in real-time inside of intact nuclei. Through development of a molecular assembly strategy, chimeric array of gRNA oligo (CARGO), and couple which to dCas9 live-cell imaging, I achieved arbitrary and non-invasive labeling of single-copy, non-repetitive DNA elements, including enhancers and promoters. With CARGO-dCas9 imaging, I performed the first direct measurement of cis-regulatory element dynamics quantitatively inside of living cells under normal cellular differentiation where cis-regulatory elements undergo changes of their activity. I discovered that the mobility of cis-regulatory elements, including enhancers and promoters, increases congruently with the transcriptional activity of the cognate genes. Mechanistically, this increase of mobility likely arises from non-thermal sources of molecular agitation. These results directly challenge the conventional wisdom of stable enhancer-promoter looping model for cis-regulation and argue for an under-appreciated biophysical aspect of cis-regulatory element dynamics inside of living cells. An array of recently developed technologies revealed the organizing principles of chromatin at sub-micron length-scale and established topologically associated domains (TADs) as the basic structural and functional unit at this length-scale. Meanwhile, trans-acting factors that are involved in regulation of TADs have also been identified, including CCCTC-binding factor (CTCF) and the cohesin complex. In the second part of my thesis, I investigated the molecular interplay of CTCF, the cohesin complex and transcription-related processes in native chromatin environment by combining advanced genetic engineering with quantitative super-resolution imaging approaches. I discovered characteristic molecular compositions of homotypic assemblies of CTCF and the cohesin complex, as well as characteristic molecular compositions and arrangements of heterotypic assemblies formed between CTCF and the cohesin complexes in single cells. Furthermore, I uncovered the interdependency between the establishment of these molecular assemblies in native chromatin environment. These results emphasize the impact of cellular environment on the behaviors of trans-acting factors regulating chromatin structures and functions. Taken together, my thesis has concerned broadly the structural and functional aspects of chromatin in mammalian system. Through interdisciplinary approaches, I provided novel first-principle insights into transcription regulation and its interplay with chromatin organization in native cellular environment. The conclusions from my thesis argue for further appreciation of the cooperativity and interdependency between the structural and functional roles of mammalian chromatin in storage and regulatable accessibility of genomic information