The role of the histone variant macroH2A in the regulation of chromatin architecture

Abstract

In the eukaryotic cell nucleus, DNA is tightly wrapped around histone complexes, forming a dynamic structure known as 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: H2A, H2B, H3 and H4. 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 including transcription and DNA repair.

MacroH2A histones are the most divergent histone variants in terms of sequence and structure when compared to its replication-coupled counterpart: 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. Differences in the sequence of the macrodomain provoke changes in the size and hydrophobicity of the macrodomain pocket. The macrodomain of macroH2A1.1 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 the precise molecular mechanism by which they act on chromatin and their role in transcriptional regulation is still a complex and 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 expression 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 includes deregulation of genes related to cellular adhesion, development, hypoxia and inflammation. This includes the upregulation of the cancer-promoting gene DKK1, which is rendered sensitive to activation in response to inflammatory TNFα signaling 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.

En el nucli eucariòtic l’ADN es troba embolicat al voltant d’un complex proteic d’histones, formant la unitat estructural bàsica de la cromatina: el nucleosoma. En contextos cel·lulars i genòmics específics, les histones que formen part del nucleosoma es poden intercanviar per variants d’histona, que difereixen en seqüència i estructura de les formes “canòniques” i proporcionen característiques específiques a la cromatina que poden afectar processos com la transcripció o la resposta a dany de l’ADN.

Les histones macroH2A són variants de la histona macroH2A que es caracteritzen per una estructura tripartida: a més d’un domini histona en N-terminal, contenen una regió connectora que sobresurt del nucli proteic del nucleosoma i posiciona de manera accessible un domini macro globular. Diversos estudis han implicat les histones macroH2A en el desenvolupament, la diferenciació cel·lular, la reprogramació i el càncer. En general, es considera que actuen com a elements estabilitzadors de l’epigenoma, però els mecanismes moleculars darrera d’aquest paper no estan ben definits.

L’objectiu de la meva tesi ha estat caracteritzar el paper de les histones macroH2A en la organització de la cromatina i la regulació. Mitjançant estudis de pèrdua de funció en cèl·lules HepG2, hem determinat que macroH2A és essencial per la organització de l’heterocromatina i en particular manté l’estructura d’elements genòmics repetitius i la seva interacció amb la lamina nuclear. També hem identificat un paper de la regió connectora, carregada positivament, en la limitació de l’expansió de la cromatina en resposta a dany de l’ADN i en el manteniment d’estructures heterocromàtiques. Finalment, l’estudi transcripcional de tumors derivats de cèl·lules HepG2 revela canvis transcripcionals dependents de macroH2A rellevants en el context del càncer. En concret, l’absència de macroH2A provoca una sobreexpressió del gen DKK1, associat a diferents tipus de càncer, i permet la seva activació davant estimulació inflamatòria amb TNFα.

En conclusió, aquest estudi revela un paper essencial de la histona macroH2A en la organització nuclear i proporciona evidències per un mecanisme molecular novell que implica la regió connectora de la proteïna. Addicionalment, l’absència de macroH2A provoca canvis transcripcionals rellevants en el context del càncer, en concret la sobreexpressió de DKK1. Proposem que l’estabilització d’estructures de cromatina per part de macroH2A pot proporcionar un mecanisme pel seu paper observat en mantenir estats transcripcionals i de diferenciació cel·lular estables.