Understanding mechanisms of heterochromatin tethering at the nuclear lamina

Project Summary / Abstract
In nearly all mammalian cells, a layer of densely compacted heterochromatin resides at the nuclear periphery
while uncompacted, transcriptionally active euchromatin resides in the nuclear interior. This peripheral
heterochromatin is tethered to the nuclear lamina, a filamentous meshwork built by the Lamin proteins.
Approximately one third of the genome is compacted and repressed within lamina-associated domains (LADs)
at the nuclear periphery. LADs maintain the stable repression of gene-poor and repeat-rich regions of the
genome, which is critical for maintaining genome integrity. In addition, many lineage-specific genes are found
within LADs, leading to the model that LADs influence cell fate by restricting expression of irrelevant genes.
Mutations to components of the lamina cause at least 15 distinct “laminopathy” syndromes that are characterized
by abnormal gene expression. In spite of the clear importance of this structure to normal cellular function and its
links to human disease, it remains a mystery how association with the nuclear lamina confers repression. The
objective of my research program is to resolve this mystery by defining how components of the lamina and
chromatin communicate to mediate gene repression. In this proposal, I lay out strategies to define how lamina
protein components tether specific loci, and to determine how these loci are selectively marked for LAD
association by chromatin modification.
The nuclear lamina is a complex assembly of Lamin proteins and lamina-associated inner nuclear membrane
(INM) proteins that varies in composition across cell types. Remarkably, either of two key proteins must be
present at the nuclear lamina to mediate peripheral tethering of heterochromatin in all mammalian cells. These
proteins are the Lamin B receptor (LBR), an INM protein, and Lamin A/C. In addition to the action of these tether
proteins, some signal must specify which genomic loci are destined to be associated with the lamina in order for
LAD formation to be specific and reproducible. Recent work has indicated a strong relationship between LAD
residence and one specific heterochromatin-associated histone modification: dimethylation on histone H3 lysine
9 (H3K9me2). These observations suggest a simple model: that Lamin A/C and LBR recognize and tether
H3K9me2-marked chromatin. However, whether LBR and Lamin A/C can recognize and bind to specific features
of chromatin remains unclear. Separately, we do not understand how H3K9me2 modification comes to be
specifically enriched on LAD-destined chromatin. We will employ structure/function studies along with cellular
chromatin tethering assays and in vitro nucleosome binding assays to define how LBR and Lamin A/C recognize
and tether loci. In parallel, we will define H3K9me2 dynamics in space and time using an acute and reversible
degron approach to interfere with methyltransferase activity. Through these efforts, we will decipher how lamina
and chromatin elements work together to direct gene repression.

Grant Number: 5R35GM142897-05
NIH Institute/Center: NIH
Principal Investigator: Abigail Buchwalter