? ADDITIONAL TOOL DEVELOPMENT OR DATA GENERATION In this Additional Tool Development or Data Generation (ATDG) module we will focus on the development of several advanced technologies that will further boost the analysis of genome compartmentalization. First, we will address the urgent need to manipulate large genomic regions in order to gain insights into the mechanisms and functions of genome compartmentalization. It is likely that the targeting of genomic loci to specific compartments is encoded in multiple sequence elements dispersed over long stretches of DNA. Identification of these targeting signals will therefore require extensive manipulation of long genomic regions. We will develop a pipeline for the custom design and automated assembly of ~100kb DNA sequences, which will enable us to freely edit, delete, or add multiple sequence elements. In addition, we will establish protocols and cell lines for the easy integration of such large DNA regions into the genome. Combined, these tools will allow for the systematic dissection of the mechanisms responsible for genome compartmentalization. Second, we will develop an extensive set of reagents for easy visualization and live-cell tracking of individual genomic loci. Using our automated DNA synthesis pipeline, we will generate a large collection of TALE probes that each bind to a single-locus tandem repeat. This will enable the in vivo microscopy of hundreds of specific genomic loci. For substantially higher sensitivity, we will develop improved genome visualization using split-GFP TALEs. Additionally, we will develop staining of fixed cells using pools of purified TALE proteins. Third, we will address the need to construct maps of genome ? compartment associations in single cells; such maps are crucial for our understanding of cell-to-cell variability of genome organization. We have recently established a modified DamID protocol that can map nuclear lamina interactions genome-wide in single cells. As this protocol has so far only been used in haploid human cells, we will adapt it for use in diploid cells, using SNPs to discriminate homologous chromosomes. We will then apply this protocol to map genome interactions with several nuclear compartments in series of single cells. These three technologies will greatly enhance our abilities to manipulate, visualize and map regions of the genome that associate with specific nuclear compartments. They will not only pave the way for advanced studies in the Biological Validation Development module and beyond, but also yield novel and unique protocols, reagents and data sets for the scientific community.
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