James M. Holaska, Ph.D.

Functional analysis of nuclear envelope proteins in transcription regulation and disease

    My long-term research interests are to elucidate the molecular mechanisms underlying nuclear envelope regulation of fundamental cellular processes including transcription, chromatin remodeling, cell signaling and DNA replication. We study proteins at the inner nuclear envelope to begin elucidating the mechanism of nuclear envelope regulation of these processes. Our lab primarily focuses on one of these inner nuclear membrane proteins, emerin, to determine how nuclear envelope proteins regulate gene expression and nuclear architecture. The three broad areas on my lab focuses are: (a) how multiple, biochemically distinct emerin complexes function to integrate the regulation of gene expression and nuclear architecture, (b) the mechanisms of emerin-regulated gene expression and (c) the role of nuclear envelope protein reorganization in mesenchymal stem cells (MSCs) during differentiation and the acquisition of tissue-specific gene expression.

The nuclear envelope is composed of two lipid bilayers, the outer nuclear membrane, which is contiguous with the endoplasmic reticulum, and the inner nuclear membrane. Positioned within the inner nuclear membrane are ~ 80 integral membrane proteins. A number of these proteins are members of the LEM-domain family of nuclear proteins, which are named for the founding members, LAP2, Emerin and MAN1. The LEM domain mediates binding to the chromatin-associated protein Barrier-to-Autointegration Factor (BAF) and thus it was proposed that binding of LEM-proteins to chromatin-bound BAF might recruit chromatin to the nuclear envelope. Stable localization of LEM-proteins is dependent on their interaction with lamin proteins, which form a nuclear envelope-associated intermediate filament network underlying the nuclear envelope.

Mutations in the genes encoding emerin and lamin A produce diseases with a broad spectrum of overlapping, but distinct, phenotypes. These diseases include progressive skeletal muscle weakening, heart muscle dysfunction, life-threatening irregular heart rhythms, contractures of major tendons, abnormal fat deposition and premature aging. Emerin and lamin A are expressed in all differentiated cell types, yet these diseases specifically affect heart, muscle, tendons and fat. To explain the tissue specificity of nuclear envelope-associated diseases, emerin and lamin A were variously proposed to have roles in tissue-specific gene expression, cell signaling or nuclear structure. Experimental evidence supports all models, but no clear molecular mechanism has emerged. Understanding the molecular mechanisms of emerin and lamin function in both gene expression and nuclear architecture, and the integration of these functions, will be crucial for understanding the mechanism of nuclear envelope diseases.

Regulation of Lmo7 transcription activity by emerin: Implications for muscular dystrophy

We identified Lmo7, a LIM-domain only protein, as a transcription activator of emerin and other muscle and cardiac genes. Lmo7-null mice have dystrophic muscles suggesting that Lmo7 is important for muscle differentiation, regeneration, or function. We also showed that emerin-binding negatively regulates Lmo7 activity. Since mutations in emerin cause Emery-Dreifuss muscular dystrophy (EDMD), we hypothesized that Lmo7 is an EDMD-relevant transcription regulator. Supporting this hypothesis Lmo7 directly binds myogenic genes and activates myogenic differentiation. To begin understanding the functional interaction between Lmo7 and emerin, and how it regulates tissue-specific gene expression crucial for muscle differentiation and function, my lab currently focuses on (a) characterizing the domain structure of Lmo7, (b) analyzing the molecular mechanisms of Lmo7-dependent transcription, and (c) investigating the regulation of myogenic differentiation by emerin and Lmo7.

The role of nuclear envelope proteins in mesenchymal stem cell (MSC) differentiation

Another hypothesis for how mutations in emerin and lamin A cause disease is that emerin and lamin A are key regulators of MSC differentiation, since MSCs differentiate to form the affected tissues in nuclear envelope-associated diseases. Thus, we propose that loss or mutation of emerin or lamin A causes defects in MSC differentiation and regeneration of the MSC-derived tissues. Experiments designed to test this hypothesis include (a) determining the expression levels of nuclear envelope proteins during MSC differentiation, (b) identifying emerin-regulated differentiation genes, (c) characterizing emerin’s regulation of MSC differentiation genes, and (d) testing if emerin regulates genomic architecture by initiating and maintaining the heterochromatic state at the nuclear envelope.  

Emerin and chromatin remodeling

Generally, expressed genes are contained within regions of decondensed chromatin structures called euchromatin. Repressed genes tend to reside in regions of compacted chromatin called heterochromatin. The position of heterochromatin is often seen at the nuclear periphery juxtaposed to the nuclear envelope and euchromatin localizes to the nuclear interior. Peripheral localization of repressed chromatin led us to hypothesize that the nuclear envelope either initiates or maintains heterochromatin formation. Since emerin interacts with chromatin-repressive machinery, we hypothesize that emerin actively represses chromatin. My lab is actively testing this hypothesis by studying (a) the formation of emerin-containing chromatin remodeling complexes at the nuclear envelope, (b) the regulation of chromatin remodeling complex formation in vitro and in vivo, and (c) the regulation of heterochromatin formation by emerin using biochemical and cell biological techniques.

Holaska, J.M. and Wilson, K.L. An emerin proteome: purification of distinct emerin-containing complexes from HeLa cells suggests molecular basis for diverse roles including gene regulation, mRNA splicing, signaling and nuclear architecture. Biochemistry, 46:8897-8908, 2007.

Holaska, J.M. and Wilson, K.L. Lmo7, an emerin-binding protein that shuttles between the cell surface and nucleus and regulates emerin transcription. Hum. Mol. Genet., 15:3459-3472, 2006.

Holaska, J.M. and Wilson, K.L. Multiple roles for emerin: implications for Emery-Dreifuss muscular dystrophy.  Anat. Rec. A. Discov. Mol. Cell. Evol. Biol., 288:676-80, 2006.

Holaska, J.M., Kowalski, A.K. and Wilson, K.L. Emerin caps the pointed end of actin filaments: evidence for an actin cortical network at the nuclear envelope. PloS. Biol., 2:1354-1362, 2004.

Holaska, J.M., Lee, K.K., Kowalski, A.K. and Wilson, K.L. Transcriptional repressor germ cell-less (GCL) and barrier to autointegration factor (BAF) compete for binding to emerin in vitro. J. Biol. Chem., 278:6969-75, 2003.