DIRECTED REORGANIZATION OF CHROMATIN TO THE NUCLEAR LAMINA IS MEDIATED BY CHROMATIN STATE, YING-YANG1 AND A-TYPE LAMINS

Nuclear Organization;Nuclear lamina;Epigenetics;Chromatin;Ying-Yang1;A-Type Lamins;Biology

Harr, Jennifer CWilson, Katherine L ;

Johns Hopkins University

关键词: Nuclear Organization;    Nuclear lamina;    Epigenetics;    Chromatin;    Ying-Yang1;    A-Type Lamins;    Biology;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60514/HARR-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Nuclear organization has been implicated in regulating gene activity. Recently, large developmentally regulated regions of the genome that are dynamically associated with the nuclear lamina have been identified. However, little is known about how these lamina-associated domains (LADs) are directed to the nuclear lamina. We have generated genome-wide data that identifies lamina associated regions in desperate cell types, to study the organization of LADs. We utilize this data in combination with the development of the tagged chromosomal insert site system (TCIS) to discern the structural and functional relationships behind nuclear organization. The TCIS system is comprised of a recombination cassette, to allow site-specific Cre-mediated recombination, as well as a lacO array that can reversibly bind EGFP-LacI for visualization of the locus. TCIS is then integrated in a single copy in a single location in fibroblast genome. Thus, TCIS enables integration of a single copy of any DNA fragment into the genome by directed recombination into the TCIS site and its visualization.Because any genomic element of interest can be specifically recombined into TCIS, this system is ideal for assaying association with a nuclear compartment of interest. This system makes it possible to study the mechanistic and functional consequences of genome organization at a single locus in a native state. In this work we utilize TCIS to investigate the structural/functional relationships of the periphery as a nuclear sub-compartment. We have generated genome-wide data produced by DNA Adenine Methyltransferase Identification (Dam-ID) to identify small sequences from the borders of fibroblast-specific variable LADs that are sufficient to target these ectopic sites to the nuclear periphery. We identified YY1 (Ying-Yang1), CTCF, BTB-POZ domain protein binding sites (amongst others) as being enriched in relocating sequences. Knockdown of YY1 or lamin A/C, but not lamin A, led to a loss of lamina association. In addition, targeted recruitment of YY1 proteins facilitated ectopic LAD formation dependent on histone H3 lysine 27 trimethylation and histone H3 lysine di- and tri-methylation. Our results also reveal that endogenous loci appear to be dependent on lamin A/C, YY1, H3K27me3, and H3K9me2/3 for maintenance of lamina-proximal positioning.