Faculty of Medicine. Settore disciplinare: BIO/10. functional domains. Monica Soldi. IFOM-IEO Campus, Milan. Matricola n. R08407

Transcription

1 PhD degree in Molecular Medicine European School of Molecular Medicine (SEMM), University of Milan and University of Naples Federico II Faculty of Medicine Settore disciplinare: BIO/10 Establishment and optimization of the ChroP approach, combining ChIP and MS-based proteomics, for the characterization of the chromatome at distinct functional domains Monica Soldi IFOM-IEO Campus, Milan Matricola n. R08407 Supervisor: Dr. Tiziana Bonaldi IFOM-IEO Campus, Milan Added co-supervisor: Dr. Gioacchino Natoli IFOM-IEO Campus, Milan Anno accademico

2 1. ABSTRACT In eukaryotes the genetic information is stored in chromatin, a highly structured nucleoprotein complex that mediates the coordinated regulation of gene expression. The basic unit of chromatin is the nucleosome, which consists of 147bp of DNA wound around a histone octamer core containing two copies of the histones H2A, H2B, H3 and H4. Changes in chromatin structure, which do not involve the nucleotide sequence, can translate into transient or heritable adjustments in gene expression. Various mechanisms modulate chromatin states: among them a pivotal role is played by covalent histone posttranslational modifications (hptms), for which the repertoires of combinations and positions are extremely varied. In addition to hptm patterns, chromatin is characterized by the local enrichment of a distinct set of histone variants; binding proteins, including various ATP-dependent chromatin remodelling complexes; DNA methylation and differential nucleosome density. Together, these components contribute to the establishment of specific chromatin landscapes, defining the functional state of the genome in that territory. Chromatin immunoprecipitation (ChIP) and Mass Spectrometry (MS) are complementary strategies to investigate the epigenetic components of chromatin. ChIP followed by deep sequencing (ChIP-Seq) allows genome-wide profiling of hptms and binders at individual genes and regulatory regions, up to a resolution of inividual nucleosomes. However, ChIP does not inform about the protein portion of chromatin, knowledge instead offered by MS-based proteomics. At the level of individual histones, MS enables to detect virtually all hptms in an unambiguous and unbiased fashion and to reveal interplays between them. Yet, MS analysis on bulk chromatin preparations limits the inspection of PTMs to a global view, with no information about their patterning in distinct functional regions. Nowadays, a global investigation of synergies between histone PTMs, 10

3 variants, and chromatin-associated proteins in a locus-specific manner remains a very attractive unachieved goal. During the course of my PhD, I contributed in this direction developing and optimizing a global, quantitative proteomic strategy, named ChroP (Chromatin Proteomics), for the analysis of the protein component of distinct chromatin regions, enriched by modified and preparative version of ChIP. I developed two ChroP protocols, which differ in the step of chromatin IP: the Native-ChIP (N-ChIP) was used to dissect histone PTM patterns whereas the Crosslinking ChIP (X-ChIP) was used in combination with SILAC-based interactomics to characterize proteins interacting with the domains of interest. I used the antibodies against tri-methylated Lysine 9 and Lysine 4 on histone H3 (H3K9me3 and H3K4me3) to enrich functionally distinct and non-overlapping chromatin regions from HeLa nuclei. High-resolution MS of the fractionated nucleosomes enabled a dissection of the domain-specific composition in terms of histone modifications, variants and non-histonic proteins, which we refer to as the modificome and interactome. First of all, I observed the expected combinatorial enrichment of silent modifications in H3K9me3, and of active ones in H3K4me3. The accordance of my results with previous studies allows to validate the robustness of the approach and, with this confidence, I could investigate novel PTMs. Remarkably, ChroP exhibited a unique and peculiar strength in revealing PTMs associations not only at the intra-molecular level within H3, but also across the different core histones, within the same nucleosome. The SILAC-based investigation of co-associated proteins revealed a number of histone variants and multi-protein complexes differentially enriched in the two functional territories. Some of them confirmed several previously described interactions, thereby validating our method. In addition, I identified numerous novel interactors, suggesting potential novel roles and regulating chromatin pathways for these proteins. A representative case was the variant H2A.X with the WICH chromatin-remodeling complex, accumulating in the H3K9me3 regions. I thus propose a higher local density model for 11

4 H2A.X in heterochromatin and provide evidence that this accumulation, together with the recruitment of WICH, represents an additional level of modulation of the DNA damage response (DDR) in this chromatin compartment. The ChroP approach is relatively easy to setup, given the limited changes made to the conventional N- and X- ChIP protocols. Hence, ChroP emerges as a potential useful tool to dissect chromatin composition and understand how all the distinct protein components can act in a concerted manner to enforce a specific chromatin status. 12

5 2. INTRODUCTION 2.1 Chromatin, epigenetics and histone post-translational modifications Chromatin is a highly ordered nucleoprotein complex that mediates both the DNA compaction into the eukaryotic nucleus and the regulation of gene expression. At the structural level, the basic unit of chromatin is the nucleosome, consisting of 147bp of DNA wound around an octamer core containing one histone H3-H4 tetramer and two histone H2A-H2B dimers (1, 2). Functionally, chromatin is organized into two distinct regions: euchromatin is less condensed and generally permissive for transcription, whereas heterochromatin is highly condensed and transcriptionally silent. Heterochromatin is classified as being either constitutive or facultative. In constitutive heterochromatin, the DNA remains condensed throughout the cell cycle. In facultative heterochromatin however the DNA can loose its condensed form and become transcriptionally active in response to distinct signals (3-5). Changes in chromatin structure that do not involve the nucleotide sequence can translate into heritable adjustments of gene expression and thus constitute an epigenetic memory system of the cell (6-10). Epigenetic inheritance can be explained through a stepwise model proposing that epigenator, initiator and maintainer factors operate sequentially and synergistically to enforce and maintain specific functional states of the genome (11). The epigenator, a signal emanating from the external environment, is translated by an initiator into a specific chromatin/dna functional state, which is sustained by a number of different maintainer factors. These include the methylation of cytosine in CpG islands (12, 13), covalent post-translational modifications of histones (hptms) and, in light of more recent studies, the activities of non-coding RNAs (ncrna) (14, 15). 13

6 Among the epigenetic maintainers listed, histone PTMs are largely recognized as key regulators of chromatin structure and function. hptms include acetylation, ubiquitination and sumoylation of Lysines; different methylation degrees of Arginines and Lysines; phosphorylation of Serines, Threonines and Tyrosines; ADP-ribosylation of Arginines, Glutamic and Aspartic acids; deimination (or citrullination) of Arginine; Proline isomerization (Figure 1) (16-18), and in addition some less-characterized modifications. Figure 1. Histone post-translational modifications. The nucleosome core particle, with the N-terminal tail of core histone and the annotation of sites of post-translational modification. Numbers along the DNA indicate each complete helical turn on either side of the dyad axis. Sites marked by green arrows are susceptible to cutting by trypsin in intact nucleosomes. Most important modifications are Acetyl Lysine (ack); methyl Lysine (mek); methyl arginine (mer); phosphoryl serine (PS); ubiquitinated lysine (uk). Adapted from Bennister A.J. and Kouzarides T. Cell Res The histone code hypothesis proposes that histone post-translational modifications act either singly or in combination to control distinct downstream pathways or processes on chromatin, ultimately defining the functional status of the underling DNA (19). The letters of this code are the modifications themselves, which are placed and removed by enzymes known as writers and erasers, respectively. hptms exert their function on chromatin through two distinct mechanisms. In the first, higher orders of chromatin structure are altered via changes in inter-nucleosomal or histone-dna interactions, thus 14

7 controlling the accessibility of DNA-binding proteins such as transcription factors (cis mechanisms). Alternatively, hptms can generate binding platforms for the recruitment of effector proteins containing specialized domains (trans mechanisms): the so-called readers of the code (Figure 2). The readers translate the information encoded by the modification patterns into specific biological outcomes (20-23). Figure 2. Domains binding modified histones. Representation of some proteins with specific domains able to specifically bind modified histones. Adapted from Kouzarides T. Cell In addition to hptm patterns, chromatin is characterized by the local enrichment of a distinct set of histone variants; binding proteins, including various ATP-dependent chromatin remodelling complexes; and differential nucleosome density and position. Together, these components contribute to the establishment of specific chromatin landscapes, defining the functional state of the genome in that territory (Figure 3) (24). 15

8 Figure 3. The distinct components contributing to define the functional state of chromatin domain. Adapted from Margueron R. and Reinberg D. Nat Rev Genet Antibodies directed against specific hptms are traditionally used to study the language of histone modification in various assays. These include: immunofluorescence (IF) analyses of modifications at the single cell level; immunoblotting (WB), which allows profiling of PTMs in different samples and/or conditions; as well as chromatin immunoprecipitation (ChIP), which can be coupled to either PCR, DNA microarray (ChIP-on-chip) or deep sequencing (ChIP-Seq) for description of their local enrichments. The last two methods allow the genome-wide mapping of modifications, with a resolution of a few nucleosomes (25-27). Antibody-based assays are hampered by limitations in their specificity and efficiency when used to reveal the combinatorial aspect of the code. In fact, modifications can occur on adjacent or closely spaced residues within the same histone, making an epitope-masking effect more likely. To address this issue, a number of strategies have been developed to assess accurately the specificity of antibodies used in chromatin research. Peach et al. combine immunoprecipitation (IP) of native HPLC-purified H3 with mass spectrometry to detect PTMs co-enriched by a certain antibody on the same polypeptide. In 16

9 addition, Fuchs et al. have developed a peptide-array assay, based on a comprehensive library of modified peptides (28, 29). Mass spectrometry (MS) has emerged as a promising complementary analytical strategy to identify known and novel PTMs on proteins, as well as for the relative quantitation and detection of interactions between them (30). The recent advent of highresolution mass spectrometry has increased the relevance of MS-based hptm analysis by enabling the discrimination of near-isobaric modifications, either singly or in combinations, on very long polypeptides and even on intact histones (31-39). Finally, the use of different labeling strategies, both chemical and metabolic, has enabled the accurate quantitation of modifications, both in a relative and absolute manner (40). The epigenomics and chromatomics fields share a common goal in studying chromatin structure, composition and features: to gain a comprehensive view, from genome to proteome, of the epigenetic phenomena underlying the establishment and inheritance of specific expression patterns (41, 42). 2.2 Mass Spectrometry analysis and MS based-proteomics The steps of a typical proteomic experiment are shown in Figure 4. Briefly, after reducing the complexity of a protein preparation by electrophoretic speparation the proteins are subjected to enzymatic digestion, tipically using trypsin as protease. After MS analysis, by which the MS informations at the peptide level are obtained, the specific proteins are identified by software-assisted database searching, with which it is also possible to identify and localize PTMs within the peptide. 17