Presentation Title

Characterizing Nucleolar Associated Heterochromatin in Mouse Cells Using 3D-FISH

Faculty Mentor

Paul D. Kaufman

Start Date

18-11-2017 10:00 AM

End Date

18-11-2017 11:00 AM

Location

BSC-Ursa Minor 35

Session

Poster 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Kevin Ou, Anastassia Vertii, Paul D. Kaufman

Department of Molecular, Cell and Cancer Biology

Characterizing Nucleolar Associated Heterochromatin in Mouse Cells Using 3D-FISH

A complete understanding of epigenetics will require knowledge about the relationships between nuclear organization and gene regulation. Recent research has shown that localization is critical for gene regulation of heterochromatin, the more condensed and transcriptionally repressed chromatin that frequently tethers to either the nuclear lamina or nucleolus. Additionally, although it is known that perinucleolar heterochromatin is particularly dynamic in developing mouse embryos, it has not been mapped systematically. Thus, we seek to map heterochromatin changes in mouse embryonic stem cells (mESC) before and during differentiation into neural progenitor cells. As a first step towards this, we developed methods for isolating, deep sequencing, and analysis of associated DNA of the nucleoli in mouse embryonic fibroblasts (MEFs). These studies revealed a new class of nucleolar associated domain (NAD) of heterochromatin that preferentially localizes to the nucleolus but not to the nuclear lamina: class II NADs. We confirmed the sequencing results by performing 3D fluorescence in situ hybridization (FISH) with class II NAD probes and quantifying their association with the nucleolus. Our results altogether provide convincing evidence of the existence of class II NADs in mouse cells and will be used to characterize NAD changes during differentiation.

This project was supported by NIH Grant U01 DA040588 as part of the 4DN Network.

Summary of research results to be presented

With the increasing amount of spotlight on epigenetics, many models have been proposed to elucidate the association between nuclear organization and gene regulation. Much of the recent ground-breaking research on chromatin organization has provided evidence that heterochromatin localization to either the nucleolus or nuclear lamina is critical for gene regulation. However, although lamina associated domains of heterchomatin (LAD) changes were observed during stem cells differentiation and were proposed to be important for cellular identity throughout differentiation, nothing is known about changes in NADs.

We therefore characterized mouse NADs composition in mouse embryonic fibroblasts (MEFs) and in mouse embryonic stem cells that undergo differentiation into neural progenitor cells. Initial characterization by deep sequencing of mouse NADs revealed two major classes of nucleolar-associated chromatin. While class I NADs demonstrate characteristics similar to LADs and are very similar to previously identified NAD characteristics in human cells, a new type of NADs, which we define as class II NADs, contains sequences that are not found in LADs during differentiation stage. From this, we speculate that in contrast to class I NADs, which are seemingly interchangeable with LADs, class II NADs are specific to the nucleolus and might require regulatory mechanisms that are different from those of LADs.

Thus, based on the sequencing analysis, BAC probes were designed and FISH experiments were performed to confirm the existence of both types of NADs in single cells. As expected, 3D-FISH results revealed the presence of both classes of NADs in association with nucleoli. For probes with low percentage of nucleolar association against successful positive controls, comparisons of their nucleolar association to NAD peak scores from bioinformatics analysis suggests a need for readjustment of threshold values of peaks in the original sequencing data, and more probes will be designed accordingly to perform further analysis with additional single cell FISH experiments. Furthermore, the NAD sequences that we confirmed with FISH will be used as target sequences for the analysis of NAD dynamics throughout differentiation of mouse embryonic stem cells. A better understanding of the similarities and differences between the mouse and human heterochromatin characteristics may reveal general rules for heterochromatic epigenetics.

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Nov 18th, 10:00 AM Nov 18th, 11:00 AM

Characterizing Nucleolar Associated Heterochromatin in Mouse Cells Using 3D-FISH

BSC-Ursa Minor 35

Kevin Ou, Anastassia Vertii, Paul D. Kaufman

Department of Molecular, Cell and Cancer Biology

Characterizing Nucleolar Associated Heterochromatin in Mouse Cells Using 3D-FISH

A complete understanding of epigenetics will require knowledge about the relationships between nuclear organization and gene regulation. Recent research has shown that localization is critical for gene regulation of heterochromatin, the more condensed and transcriptionally repressed chromatin that frequently tethers to either the nuclear lamina or nucleolus. Additionally, although it is known that perinucleolar heterochromatin is particularly dynamic in developing mouse embryos, it has not been mapped systematically. Thus, we seek to map heterochromatin changes in mouse embryonic stem cells (mESC) before and during differentiation into neural progenitor cells. As a first step towards this, we developed methods for isolating, deep sequencing, and analysis of associated DNA of the nucleoli in mouse embryonic fibroblasts (MEFs). These studies revealed a new class of nucleolar associated domain (NAD) of heterochromatin that preferentially localizes to the nucleolus but not to the nuclear lamina: class II NADs. We confirmed the sequencing results by performing 3D fluorescence in situ hybridization (FISH) with class II NAD probes and quantifying their association with the nucleolus. Our results altogether provide convincing evidence of the existence of class II NADs in mouse cells and will be used to characterize NAD changes during differentiation.

This project was supported by NIH Grant U01 DA040588 as part of the 4DN Network.