Resumen de The role of histone variant macroh2a in the regulation of chromatin architecture
In the eukaryotic cell nucleus, DNA is tightly wrapped around histone complexes, forming the chromatin. The nucleosome particle is the basic repeating unit of the chromatin fiber and its protein core is composed of the so-called core histones. Depending on the cellular and genomic context, replication-coupled histones can be exchanged by histone variants. The bulk of the cellular histone pool is composed of "canonical" or replication-coupled histones, but a smaller fraction is constituted by histone variants, that diverge to different extents in their primary sequence from their replication-coupled counterparts. The replacement of replication-coupled core histones by histone variants provides chromatin with specific characteristics and can influence all functions occurring on the chromatin template.
MacroH2A histones are variants of the histone H2A. In addition to an N-terminal histone fold, they contain a highly basic and unstructured linker region and a large macro domain in the C-terminus. The linker protrudes outside of the compact histone core of the nucleosome placing the macro domain in a very accessible position. There are three different macroH2A proteins in vertebrates: macroH2A1.1, macroH2A1.2 and macroH2A2. The macrodomain of macroH2A1.1, exclusively, is able to bind and, in some contexts, inhibit the activity of the enzyme poly-ADP-ribose polymerase 1 (PARP1).
Multiple studies have implicated macroH2A proteins in development, cellular differentiation, somatic cell reprogramming and cancer. In general, macroH2A histones are considered to be stabilizing epigenetic factors that correlate with differentiated states, but their molecular mechanism on chromatin and their role in transcriptional regulation is still an incomplete picture.
The aim of my PhD has been to characterize the role of the group of macroH2A histone variants in chromatin organization and transcription. In the first part of this study, we studied the role of macroH2A in the nuclear organization of chromatin in HepG2 cells, revealing that macroH2A removal results in a global loss of heterochromatin organization. We found that a fraction of macroH2A is enriched in H3K9me3-marked heterochromatin and is necessary for maintaining the higher-order organization of repetitive elements and their attachment to lamin B1.
In the second part of this study, we aimed to characterize the molecular mechanisms and domain requirements of the function of macroH2A in chromatin dynamics and organization. All macroH2As can suppress DNA damage-induced chromatin expansion. MacroH2A1.1 has the strongest effect which reflects its capacity to inhibit PARP1, while the highly basic linker region of all macroH2As can limit chromatin expansion to a lesser degree in a PARP1-independent manner. Moreover, the macroH2A2 linker is essential and sufficient to maintain H3K9me3-marked heterochromatin architecture.
Finally, we wanted to describe the changes in the transcription and phenotype of cells in a cancer model that depend on the presence of macroH2A. HepG2 cells lacking macroH2A have an increased colony formation and migratory capacity. Transcriptomic profiling of tumors derived from HepG2 cells shows significant gene expression changes that depend on macroH2A, which include deregulation of genes related to cellular adhesion, development, hypoxia and inflammation, and the upregulation of the cancer-promoting gene DKK1, which is rendered sensitive to activation in response to TNFα in the absence of macroH2A.
In conclusion, our results identified a major function for macroH2A in heterochromatin organization and identified two distinct mechanisms by which macroH2A histones can affect chromatin dynamics. In addition, loss of macroH2A results in transcriptional changes relevant in a cancer context. We suggest that stabilization of chromatin structures by macroH2A could provide a mechanism to explain its contribution to stable cell states.
