The group focuses on epigenetic codes and stochastic cellular processes, two themes that are currently at the crossroads of modern Cell, Molecular and Developmental Biology.
Distinguishing between information-based and structure-based epigenetic codes
Chromatin is a polymorphic assembly, manifesting itself as loosely packed euchromatin, compacted heterochromatin and highly condensed mitotic chromatin. Along with these configurations there are a myriad of subtly deferring “chromatin states” that co-exist within the boundaries of the same nucleus and are sometimes difficult to describe in conventional, cytological terms.
Apparently, the dynamic and highly variable structure of chromatin reflects its function as a coding template and serves the need to replicate and then divide the genome as faithfully as possible during the cell cycle. This clearly requires a “golden ratio” of precise control and enough flexibility at the level of chromatin folding.
Like protein folding, chromatin folding occurs in a hierarchical manner and is in part spontaneous and in part assisted. Of all factors that play a role in this complex process, the posttranslational modifications of the histone proteins deserve a special mention. Histones represent the building blocks of the nucleosome core particle and their chemical alteration, be it addition of charge, increase of hydrophobicity, or creation of new binding sites, is expected to affect the structural and functional properties of chromatin in several ways. And, since the combinatorial repertoire of histone modifications could yield millions of patterns, it would be logical to suspect that these alterations serve a “coding” or “ciphering” function.
Precisely this idea, i.e., the existence of a “histone code” that overrides the genetic code, has been proposed several years ago and dominated the literature ever since. However, it is still not clear whether histone modifications represent signals “read” and “de-ciphered” by specific effector proteins, or whether they affect directly the physical chemistry of chromatin. In other words, we do not know whether the “histone code” is an information-based, or a structure-based, code. One way to distinguish between these two alternatives is to investigate whether the mere modification of the core histones could cause a change of chromatin state, or whether it would always be necessary to invoke “readers” and “translators”, which mediate this process. Our lab tackles this problem studying posttranslational modifications that relate to chromosome condensation during mitosis and chromatin folding during replication.
One of the epigenetic marks that we have recently analyzed is a peculiar combinatorial modification established in the onset of mitosis and erased in the end of cell division. This mark involves simultaneous phosphorylation of threonine-3, trimethylation of lysine-4 and asymmetric dimethylation of arginine-8 in the amino-terminal tail of histone H3 (PMM signature). Observations made at the cellular and sub-cellular level have suggested that PMM-containing chromatin is organized in a particular fashion along mitotic chromosomes, interlinking non-contiguous domains of chromatin and resulting in the formation of transient stem-and-loop structures that function at the metaphase-anaphase checkpoint. This scenario has now been examined in detail by state-of-the-art molecular dynamics at the ms scale, in collaboration with the team of A. Politou and E. Kaxiras (Harvard University). Consistent with the idea of a structure-based code, PMM modifications allow tight “locking” of H3 tails and formation of energetically favorable complexes that can cross-link chromatin domains along mitotic chromosomes.
Occurrence of the combinatorial modification PMM in mitotic cells 9upper panel) and in stretched chromatin fibers (lower panel). The samples have been stained with specific anti-PMM (aPMM) or anti-H3-phospho-theonine 3 (aP3) antibodies and propidium iodide (pi). Note the decoration of the centromeric regions at metaphase and the quasi-periodic staining of the unraveled chromosome strands.
(published work of Y. Markaki, A. Christogianni, A. S. Politou and S.D. Georgatos)
We want to carry our previous observations further investigating: (i) the physical properties and the 3-D structure of PMM-bearing histone H3 and nucleosomes (in collaboration with A. Politou); (ii) the functional involvement of PMM modifications in the process of asymmetric cell division and chromatid segregation (in collaboration with Z. Lygerou, University of Patras); and (iii) the role of enzymes that mediate the formation of PMM during nuclear reprogramming and stem cell differentiation. Taken together, these three approaches could cover all possible levels of epigenetic regulation from the most elementary (histone-histone and nucleosome-nucleosome interactions), to the more complicated (cell division) and the most integrated and biologically relevant (self-renewal and differentiation of pluripotent cells).
Stochastic cellular processes
Signal transduction, membrane trafficking, transport of macromolecules across the nuclear envelope and transcription of eukaryotic genes have always been considered to be rapid, efficient and highly directional (i.e., vectorial) processes. However, recent observations suggest that many –if not all- cellular functions exhibit an aspect of stochasticity, i.e., randomness in the time domain. Thus, “back-and-forth” transitions, “hesitant” translocations, abrupt changes in pace and rhythm, “poised states” and cycles of modification/de-modification seem to be the rule when we look more closely in the ways the cell “breaths” and “decides” during differentiation, adaptation to a new environment and programmed death.
We have recently studied the dynamics of the integral nuclear envelope protein LBR in conventional cultures and mouse ES cells using FRAP and high-resolution light microscopy. Consistent with the ideas described above, we have documented that LBR exchange exhibits dramatic fluctuations along the contour of the same nuclear envelope, perhaps reflecting the asymmetries and the local variations in the composition of peripheral heterochromatin, nuclear lamina, nuclear pore complexes and nuclear membranes. That membrane protein dynamics may vary from site to site at steady-state is a new concept that can be theoretically extended to other non-symmetrical organelles participating in endocytosis, exocytosis and membrane remodeling. Furthermore, this stochastic behavior provides a new conceptual framework for analyzing mutations in LBR and other membrane proteins that cause animal and human disease. For this reason, we are now utilizing the ES cell platform to investigate in detail the effects of the icj and the Greenberg mutations that result in truncated forms of LBR and are known cause ichthiosis and Greenberg’s dysplasia, respectively.
Expression of mutated LBR in cultured cells. The image shows Hela cells transfected with the Greenberg-LBR mutant (Gr, green) and counter-stained with anti-Lamin B antibodies (LMB, red).
Note the typical lesions developing in these specimens, i.e., the cytoplasmic accumulations of LBR and the aggregates of lamin B.
(unpublished work by I. Yiannios and S.D. Georgatos)
Emerging information suggests that the transcriptional activity of “pluripotency genes” (Nanog, Oct4-Sox2) fluctuates in a stochastic fashion. However, a key question that remains unanswered is whether the fluctuations of upstream regulatory factors are functionally ”productive”, or just too transient or small in scale for triggering downstream effects. To explore this problem, we have joined forces with the laboratories of D. Thanos and G. Thireos (IBEEA) and the team of T. Fotsis-C. Murphy (BRI). This scientific network, that focuses on stochastic processes during reprogramming and stem cell differentiation, will investigate -among other things- chromatin and nuclear envelope dynamics in low and high-Nanog subpopulations of ES and iPS cells, employing as “reporters” heterochromatin protein 1 (HP1) and LBR. FRAP assays, multi-dimensional and super-resolution light microscopy will be utilized to distinguish between three different mechanistic scenarios: (i) that the levels of Nanog in each cell are proportional to the dynamicity of nuclear structures (no thresholds); (ii) that nuclear dynamics in Nanog-fluctuating cells change abruptly in an all-or-none fashion (existence of specific thresholds); and (iii) that Nanog fluctuations, no matter how great in amplitude and long in duration, do not correlate with changes in nuclear dynamics.