Final Report Summary - HISTONEDDR (ROLE OF HISTONE MODIFICATIONS IN DNA DAMAGE RESPONSE IN MAMMALS)
Executive Summary:
Chromatin is composed of the DNA that carries the genetic information along with associated proteins such as histones. Although initially thought of as mere structural components of chromatin, it is clear now that the proteins in chromatin have key roles in the regulation of DNA metabolism, including replication, transcription and repair. Posttranslational modifications of histones have been widely studied and constitute one of the key epigenetic mechanisms that modulate the metabolism of DNA without affecting its sequence. It is essential to understand how these marks on the histones are placed and their functional consequences through the recruitment of specific readers or the modulation of processes such as transcription or repair.
In this context, the main objectives of this project aim to understand the role of one of the readers of histone modifications, SCML2, and to dissect the mechanism controlling the deposition of the methylation in histone H3 lysine 56 (H3K56). SCML2 is one of the homologues of Sex Comb on Midleg (SCM) in Drosophila, a member of the Polycomb family of proteins. Polycomb group proteins are key developmental regulators that control the patterning of the embryo through the repression of homeotic genes. They usually work in complexes of which Polycomb Repressive Complex 1 and 2 are the best well studied. SCM is associated to PRC1 in flies, although the exact function of SCM or its mammalian homologues is not well understood.
SCML2 has two MBT repeats that mediate the interaction with monomethylated histone H4 lysine 20 or histone H3 lysine 79. These modifications have been related to the DNA damage response and therefore we decided to analyse the function of this protein in cells. We uncovered the presence of two different isoforms of SCML2, differing in the C-terminal region, where the domain (SPM) responsible for the interaction with PRC1 is located. The presence of an isoform without the SPM domain raised the question of whether both versions of the protein could have independent roles. In fact SCML2A and SCML2B have different subnuclear localization, and only SCML2A, with the SPM domain, is associated to chromatin.
Our analysis of the two isoforms of SCML2 revealed that they do have independent functions. We have demonstrated three different roles for SCML2: first, SCML2B regulates the cell cycle progression through direct contact with CDK2/CYCE and p21 complexes; second, SCML2A cooperates with PRC1 in gene repression and is targeted to target genes through the interaction with non-coding RNA, methylated lysines on histones, and PRC1; third, both isoforms interact with USP7, and this protein deubiquitinase is essential for the stability of key components of PRC1 complexes.
Briefly, SCML2B is necessary for the stabilization of p21 during early G1, fostering the efficient inhibition of accumulating CDK2/CYCE complexes that limit the progression into S phase. In turn, CDK1 and CDK2 phosphorylate SCML2 in several residues. The levels of phosphorylation of SCML2 change throughout the cell cycle and may determine the inhibition exerted by SCML2B on CDK2/CYCE complexes. This is the first time that a direct interaction between the Polycomb system and the cell cycle machinery has been detected in mammals.
Regarding SCML2A, we have discovered that it possesses an RNA binding region that is essential for its localization to chromatin. Deletion of this region as well as mutation of the MBT domains impairs the binding to target genes to different extents. SCML2A interacts with PRC1 as expected (SCML2B does not) and it colocalizes on target genes and contributes to gene repression. Thus, we propose that SCML2A uses a combination of RNA binding, methylated lysine recognition and PRC1 interaction to find the genes that need to be repressed. We have identified non-coding RNA bound by SCML2 in cells, and their specific contribution to SCML2 targeting will be investigated further.
Finally, we have detected a very strong interaction of SCML2 with USP7. This protein deubiquitinase is best known for its role in the regulation of p53 stability. Our results show that SCML2 is necessary to bring USP7 to canonical PRC1 complexes, homologous to the Drosophila version of PRC1. We have extended the analysis of the function of USP7 showing that non-canonical PRC1 complexes are also regulated by the direct interaction of USP7 with RING1B, their core catalytic subunit. Inhibition of USP7 leads to destabilization of key proteins in these complexes and a reduced PRC1 activity.
As a whole these results provide important information to understand how the repression by PRC1 works and is regulated, and its potential relationship to the cell cycle. Both PRC1 and USP7 are potential therapeutic targets in disease such as cancer and have prominent roles in normal aging. The data presented here is thus potentially relevant for both normal and pathological physiology.
Regarding H3K56 methylation, it has been shown that acetylation of this residue is essential for normal replication and chromatin assembly after DNA damage. The presence of trimethylated H3K56 was previously detected by mass spectrometry and now we have developed an antibody to measure this modification in cells, showing that this methylation does not change during the cell cycle or after DNA damage. We tried to identify the methyltransferase that mediates this modification through different approaches. Our results suggest that several enzymes contribute to the methylation of H3K56, including members of the SMYD and MLL families. Although we did not have the time to further analyse the candidates, our work has set the grounds for future studies. Additionally, the methodologies developed in this project can be applied for the analysis of other modifications that may be of interest.
In summary, we have carried out a project that dissects different aspects of the biology of histone modifications. First, the analysis of a chromatin reader, SCML2, has given insight into the relationship between chromatin and the cell cycle, as well as the regulation of PRC1 stability and function. Second, we have addressed the potential functions of a histone modification, the methylation of H3K56, trying to identify the methyltrasnferase responsible for the deposition of this mark. Our results have made a relevant contribution to the knowledge of the basic biology of chromatin with potential implications in aging and disease.