Chromatin Conformation in Development and Disease.

Chromatin domains and loops are important elements of chromatin structure and dynamics, but much remains to be learned about their exact biological role and nature. Topological associated domains and functional loops are key to gene expression and hold the answer to many questions regarding developmental decisions and diseases. Here, we discuss new findings, which have linked chromatin conformation with development, differentiation and diseases and hypothesized on various models while integrating all recent findings on how chromatin architecture affects gene expression during development, evolution and disease.

Low Input Targeted Chromatin Capture (Low-T2C).

Targeted chromatin capture (T2C) is a 3C-based method and is used to study the 3D chromatin organization, interactomes and structural changes associated with gene regulation, progression through the cell cycle, and cell survival and development. Low input targeted chromatin capture (low-T2C) is an optimized version of the T2C protocol for low numbers of cells. Here, we describe the protocol for low-T2C, including all experimental steps and bioinformatics tools in detail.

Interplay between FLI-1 and the LDB1 complex in murine erythroleukemia cells and during megakaryopoiesis.

Transcription factors are key players in a broad range of cellular processes such as cell-fate decision. Understanding how they act to control these processes is of critical importance for therapy purposes. FLI-1 controls several hematopoietic lineage differentiation including megakaryopoiesis and erythropoiesis. Its aberrant expression is often observed in cancer and is associated with poor prognosis. We showed that FLI-1 interacts with the LDB1 complex, which also plays critical roles in erythropoiesis and megakaryopoiesis. In this study, we aimed to unravel how FLI-1 and the LDB1 complex act together in murine erythroleukemia cells and in megakaryocyte. Combining omics techniques, we show that FLI-1 enables the recruitment of the LDB1 complex to regulatory sequences of megakaryocytic genes and to enhancers. We show as well for the first time that FLI-1 is able to modulate the 3D chromatin organization by promoting chromatin looping between enhancers and promoters most likely through the LDB1 complex.

Targeted chromatin conformation analysis identifies novel distal neural enhancers of ZEB2 in pluripotent stem cell differentiation.

The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat-Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2's gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons.