Ph.D. Genetics, Universidad Autónoma de Barcelona, Barcelona (Spain)

Chromatin structure and Function

The long-term goal of my laboratory is to elucidate how chromatin organization mediates genome function in eukaryotic cells. In particular, we are interested in understanding the role of insulator proteins in the tridimensional organization of the genome in the nucleus and the significance of such organization in nuclear processes. Insulator proteins function by stabilizing contacts between distant sites along the chromatin fiber, facilitating the formation of chromatin loops. Because of this ability it is widely accepted that insulator function is required for orchestrating gene transcription throughout the genome during embryo development and during cell differentiation. Indeed, insulator function is highly conserved throughout eukaryotes, including yeasts, worms, insects and vertebrates as well as humans.

The physiological properties of insulator proteins, however, are only defined based on their ability to block enhancer-promoter interactions and to block heterochromatin spread in transgenic assays, and although a large body of chromatin immunoprecipitation data describing the distribution of insulator sites in eukaryotic genomes generally agrees with the interpretation that insulators help partitioning the genome into transcriptional domains, evidence supporting that the fundamental function of chromatin insulators is transcription regulation through the establishment of higher order chromatin structures is only indirect, and a direct mechanistic interpretation of insulator properties and function is still lacking.

One key evidence supporting the notion that insulators organize genomes into chromatin domains came first from the observation in Drosophila that insulator proteins form dense foci associated to chromatin. These foci are prominent macromolecular structures detected within the nucleus during interphase in the cell cycle, formed by the coalescence of multiple insulator proteins. Based on the insulator properties mentioned above it was suggested that these foci, also termed insulator bodies, were bound to chromatin and were the reflection of a global genome organization mediated by insulators as they arrange chromatin fibers into a three dimensional web of contacts between distant genome sites.

While the fundamental model depicting insulators as structural proteins that facilitate the tridimensional organization of chromatin in the nucleus is still supported by multiple sources of evidence, experiments in my laboratory have revealed that insulator bodies correspond in fact to stress response bodies. Our work has demonstrated that insulator bodies consist of macromolecular structures formed by the coalescence of all known insulator proteins, and that they are not present during normal S phase. Instead, we have shown that insulator bodies form exclusively in response to osmotic stress and during cell death. We have characterized this stress response and have shown that after osmotic stress insulator proteins largely detach from chromatin to assemble into insulator bodies. Insulator proteins can also disassemble from insulator bodies, repopulating chromatin by regaining binding to DNA insulator sites after recovery of isotonic conditions. Our results also demonstrate that the formation of insulator bodies in response to osmotic stress is independent of the High Osmolarity Glycerol (HOG1)–p38 Mitogen-Activated Protein Kinase pathway, underscoring that the functional meaning of this response to osmotic stress remains largely unknown.

The significance of the insulator response to osmotic stress is emphasized by the fact that removing insulator proteins from chromatin may have vast effects in higher order chromatin organization in the nucleus, since insulators under stress conditions no longer support long-range chromatin interactions. This response does not impair the ability of the genome to regulate transcription given that hundreds of genes are activated by the p38 Mitogen-Activated Protein Kinase pathway during the stress response, emphasizing that the structural and physiological consequences of insulator-dependent restructuring of chromatin organization during osmotic stress are poorly understood.

Current projects in my laboratory address fundamental questions of chromatin insulators function, genome organization, gene transcription regulation and cell cycle in eukaryotes using the osmotic stress response and Drosophila and vertebrate cell lines as a model system. To address these questions we are utilizing a variety of techniques aimed at performing in vivo functional analyses. We combine genetic analysis with an important toolkit of modern molecular biology and microscopy techniques, including RNA interference, fluorescence microscopy, live imaging, molecular cloning, DNA sequencing, PCR, Real Time-PCR, Western blot, Protein purification, Cell culture, yeast two-hybrid, Protein immunoprecipitation, Chromatin Immunoprecipitation and Chromosome Conformation Capture, among others. These techniques allow us to address mechanistic questions in vivo and at the molecular level.

Schoborg T. Rickels R, Barrios J, And Labrador M. (2013) Chromatin insulator bodies are nuclear structures that form in response to osmotic stress and cell death. The Journal of Cell Biology. 2013 Jul 22;202(2):261-76.

Schoborg TA, Kuruganti S, Rickels R, And Labrador M. (2013) The Drosophila gypsy Insulator Supports Transvection in the Presence of the vestigial Enhancer. Plos ONE. 2013 Nov 13;8(11):e81331

Wallace HA, Plata MP, Kang HJ, Ross M, Labrador M. (2010) Chromatin insulators specifically associate with different levels of higher order chromatin organization in Drosophila. Chromosoma. Apr;119(2):177-94. Epub 2009 Dec 23.

Schoborg TA, Labrador M. (2010) The phylogenetic distribution of non-CTCF protein insulators is limited to insects and reveals that insulator protein BEAF-32 is Drosophila lineage specific. Journal of Molecular evolution. Jan;70(1):74-84. Epub 2009 Dec 19

Plata MP, Kang HJ, Zhang S, Kuruganti S, Hsu SJ, Labrador M. (2009) Changes in chromatin structure correlate with transcriptional activity of nucleolar rDNA in polytene chromosomes. Chromosoma. Jun;118(3):303-22. Epub 2008 Dec 9

Labrador M, Sha K, Li A, Corces VG. (2008) Insulator and Ovo proteins determine the frequency and specificity of insertion of the gypsy retrotransposon in Drosophila melanogaster. Genetics. Nov;180(3):1367-78.