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The Wistar Institute

3601 Spruce Street

Philadelphia, PA 19104

 

Office: 215-898-3933

Research

Deciphering three-dimensional genome structure

 

Modeled 3D structure of the fission yeast genomeRecent technological advancements have allowed us to begin to model complex 3D genome structures in the nucleus. We have modeled the 3D genome structure of the model organism fission yeast using a genomic approach that combines the molecular biology procedure called chromosome conformation capture (3C) and next-generation DNA sequencing. To accurately model this genome structure, we successfully fused microscopy and genomic data.

[In the figure: Modeled 3D structure of the fission yeast genome.]

We showed that distinct chromosomal territories exist in fission yeast and demonstrated that highly transcribed genes, co-regulated genes, and functionally related genes tend to co-localize in the in vivo genome structure. We also identified conserved DNA motifs present at gene promoter regions, likely recognized by transcription factors, which play a role in facilitating those gene associations. We found that our simple model organism, fission yeast, has a functional genome organization similar to the mammalian transcription factories.

This project is supported by The NIH Director’s New Innovator Award and The G. Harold & Leila Y. Mathers Foundation.


 

Pol III gene-mediated genome organization

 

We have previously shown that the RNA polymerase III (Pol III) transcription factor complex, TFIIIC, participates in organizing the higher-order genome structure in fission yeast. We have recently found that Pol III transcribed genes can localize to centromeres and contribute to a global genome organization in interphase. We also showed that the centromeric association of Pol III genes is mediated by the condensin complex, which is known to function in chromosome condensation during mitosis, and demonstrated that the centromeric association of Pol III genes participates in chromosome condensation during mitosis. An understanding of the structure of mitotic chromosomes has long been elusive, but our study has now partially resolved this long-standing question.

This project is supported by The V foundation and The Edward Mallinckrodt, Jr. Foundation.



 Centromeric association of dispersed Pol III genes[In the figure: A number of Pol III genes such as tRNA and 5S rRNA genes dispersed throughout the fission yeast genome associate with centromeres. This centromeric association of Pol III genes is mediated by Pol III transcription machinery and condensin complex (top). The centromeric association of Pol III genes likely influences global higher-order chromosome structure, helping mediate formation of numerous chromatin loops derived from centromeres (middle). In mitosis, the centromeric association of Pol III genes contributes to chromosome condensation essential for faithful chromosome segregation (bottom).]
 




Retrotransposon-mediated genome organization

 

We have recently demonstrated involvement of Ku in both telomere tethering to the nuclear periphery and clustering of retrotransposons at centromeres in fission yeast (Tanaka et al. 2012). We showed that clustering of retrotransposons at centromeres involves Ku, condensin, and the CENP-B factor, Abp1. Intriguingly, histone H3K56 acetylation, which is known to function in DNA replication and repair, interferes with the binding of Ku and condensin to retrotransposons, thereby releasing condensin-mediated genome organization during S phase and upon DNA damage. Upon DNA damage, ATR kinase mediates the degradation of the Hst4 HDAC specific to H3K56, leading to a DNA damage-response of condensin-mediated genome organization through H3K56 acetylation. Interestingly, clustering of retrotransposons at centromeres is concerned with the efficient degradation of Hst4 upon DNA damage. In addition, our study suggests that Ku localization becomes diffuse upon DNA damage through H3K56 acetylation, and this diffusion of Ku probably facilitates the nonhomologous end joining (NHEJ) process. We also showed that H3K56 acetylation participates in telomere tethering to the nuclear periphery, indicating a global role for this specific histone modification in genome organization.

This project is supported by The G. Harold & Leila Y. Mathers Foundation.

 

[In the figure: (A) A schematic model for retrotransposon-mediated genome organization.  The CENP-B subunit Abp1 binds to Tf retrotransposons and recruits Ku, which in turn loads the genome-organizing machinery condensin onto chromatin. Condensin associating with retrotransposons and centromeres mediates retrotransposon clustering at centromeres. (B) The epigenetic mechanism that regulates Ku and condensin binding to retrotransposons.  H3K56 acetylation releases Ku and condensin from retrotransposons and, thereby, disassembles this genome organization.]

 

Visualizing associations between discrete genomic regions in live cells
 

We recently established a live-cell imaging system in our lab. We first integrated lacO repeats into the genomic region of interest and introduced LacI-GFP proteins by genetic crosses, which allows us to visualize the genomic region carrying lacO repeats. Using this methodology, we were able to visualize several diverse genomic regions: centromeres, telomeres, ribosomal DNA (rDNA) repeat locus, and loci carrying retrotransposons or Pol III-transcribed genes including tRNA and 5S rRNA, in live fission yeast cells. Using a laser scanning confocal microscope, we examined positions of the genomic loci in the 3D nuclear compartment at as short as 1.5-second intervals. Importantly, these unprecedented studies to elucidate genomic associations in the 3D nuclei of live cells demonstrated that associations between centromeres and the genomic loci carrying Pol III-transcribed genes or retrotransposons are highly dynamic. Our results revealed that centromeric motion, which is controlled by cytoplasmic microtubules, contributes to the mobility of non-centromeric genomic loci. We proposed that centromeres serve as “genome flexibility elements” by connecting highly mobile centromeres to dispersed genomic regions (Kim et al. 2013).

Stuff

This project is supported by The G. Harold & Leila Y. Mathers Foundation.

[In the figure: A model for the role of centromeric motion in the mobility of interphase genomic loci. Microtubule polymerization in cytoplasm actively pushes centromeres in nucleoplasm. Centromeres associate with dispersed genomic loci. These associations are mediated by condensin. Centromeres and their associating chromosomal loci migrate in a coordinated fashion.]