Introduction

The laboratory of Alexey Ivanov, Ph.D., focuses on mechanisms that regulate gene expression with a special emphasis on how the DNA-packaging structure of chromatin is regulated during cellular processes. The laboratory seeks to define the biochemical and molecular mechanisms that govern the normal silencing of genes during development and homeostasis, as well as disruptions of these governing mechanisms during tumor initiation and progression.

Research Summary

Eukaryotic genomes are, in general, in a default state of repression, where the vast majority of genes are turned off or silenced. This repression is accomplished largely through packaging DNA into tightly coiled DNA-protein fibers called chromatin by association with histones and other proteins. This means that chromatin must be uncoiled before allowing genes to be accessed by proteins that mediate transcription. One mechanism that regulates chromatin structure is the attachment of chemical groups to the histones. It is now becoming clear that a diverse array of enzymes modify histones and place a range of various chemical modifications, including acetylation, phosphorylation, methylation, and sumoylation. One appealing idea is that the pattern and identity of histone modifications constitute a "code" for specific processes, such as transcription. The histone code is written in proteins, not DNA, it can be heritable and constitutes the basis of epigenetics which is increasingly recognized as an important factor in cancer development.

The main system for studying the mechanisms of transcriptional repression in the lab is KRAB domain containing transcription factors. The KRAB-Zinc finger (KRAB-ZNF) superfamily of DNA-binding transcriptional repressors is the largest family of gene silencers encoded in the human genome: Estimates are that of the >700 Cys2-His2 class zinc finger genes, more than 400 contain the highly conserved ~75 amino acid KRAB repression domain. KRAB-ZNF mediated transcriptional silencing requires a direct, high affinity interaction with an obligate co-repressor KAP1 which serves as a scaffolding protein for recruitment of repression machinery. The C-terminus of KAP1 contains an HP1 interaction domain, a PHD domain and a Bromodomain, all of which are required for optimal repression.

We identified that silencing by the KRAB-ZNF-KAP1 system requires post-translational modification of KAP1 by SUMO, the 17 kDa ubiquitin-like molecule, on specific lysines in the Bromodomain. The adjacent PHD domain serves as an intramolecular E3 ligase for Bromodomain sumoylation. The attached SUMO moiety is recognized by the histone deacetylase complex NuRD and the histone methyltransferase SETDB1. These proteins place chromatin repressive marks on histones and help to establish silenced state of KRAB-ZNF target genes.

The SUMO modification of proteins in general is required for normal chromosome condensation and mitosis. Disruption of sumoylation pathway leads to mitotic defects and embryonic development failure characterized by inability of cells to properly condense chromatin. We pursue a hypothesis that mitotic chromatin condensation and heterochromatic gene silencing are intrinsically linked at the biochemical level, both being dependent on SUMO modification of structural proteins and enzymatic machinery shared by these processes.