Tiziana Bonaldi

Tet Proteins Connect the O-Linked N-acetylglucosamine Transferase Ogt to Chromatin in Embryonic Stem Cells

O-linked N-acetylglucosamine (O-GlcNAc) transferase (Ogt) activity is essential for embryonic stem cell (ESC) viability and mouse development. Ogt is present both in the cytoplasm and the nucleus of different cell types and catalyzes serine and threonine glycosylation. We have characterized the biochemical features of nuclear Ogt and identified the ten-eleven translocation (TET) proteins Tet1 and Tet2 as stable partners of Ogt in the nucleus of ESCs. We show at a genome-wide level that Ogt preferentially associates with Tet1 to genes promoters in close proximity of CpG-rich transcription start sites. These regions are characterized by low levels of DNA modification, suggesting a link between Tet1 and Ogt activities in regulating CpG island methylation. Finally, we show that Tet1 is required for binding of Ogt to chromatin affecting Tet1 activity. Taken together, our data characterize how O-GlcNAcylation is recruited to chromatin and interacts with the activity of 5-methylcytosine hydroxylases.

Polycomb-Dependent H3K27me1 and H3K27me2 Regulate Active Transcription and Enhancer Fidelity

H3K27me3 is deposited at promoters by the preferential association of Polycomb repressive complex 2 (PRC2) with CpG-rich DNA elements regulating development by repressing gene transcription. H3K27 is also present in mono- and dimethylated states; however, the functional roles of H3K27me1 and H3K27me2 deposition remain poorly characterized. Here, we show that PRC2 activity is not only associated with H3K27me3 but also regulates all forms of H3K27 methylation in a spatially defined manner, contributing to different genomic functions in mouse embryonic stem cells. H3K27me1 accumulates within transcribed genes, promotes transcription, and is regulated by Setd2-dependent H3K36me3 deposition. Contrarily, H3K27me2 is present on approximately 70% of total histone H3 and is distributed in large chromatin domains, exerting protective functions by preventing firing of non-cell-type-specific enhancers. Considering that only 5%-10% of deregulated genes in PRC2-deficient cells are direct H3K27me3 targets, our data support an active role for all H3K27 methylated forms in regulating transcription and determining cell identity.

The HP1α-CAF1-SetDB1-containing complex provides H3K9me1 for Suv39-mediated K9me3 in pericentric heterochromatin

Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1α (HP1α)–chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non‐nucleosomal histone H3. Therefore, the heterochromatic HP1α–CAF1–SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1α with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.

Mass spectrometry-based identification and characterisation of lysine and arginine methylation in the human proteome

Protein methylation is a post-translational modification (PTM) by which a variable number of methyl groups are transferred to lysine and arginine residues within proteins. Despite increased interest in this modification due to its reversible nature and its emerging role in a diverse set of biological pathways beyond chromatin, global identification of protein methylation has remained an unachieved goal. To characterise sites of lysine and arginine methylation beyond histones, we employed an approach that combines heavy methyl stable isotope labelling by amino acids in cell culture (hmSILAC) with high-resolution mass spectrometry-based proteomics. Through a broad evaluation of immuno-affinity enrichment and the application of two classical protein separation techniques prior to mass spectrometry, to nucleosolic and cytosolic fractions separately, we identified a total of 501 different methylation types, on 397 distinct lysine and arginine sites, present on 139 unique proteins. Our results considerably extend the number of known in vivo methylation sites and indicate their significant presence on several protein complexes involved at all stages of gene expression, from chromatin remodelling and transcription to splicing and translation. In addition, we describe the potential of the hmSILAC approach for accurate relative quantification of methylation levels between distinct functional states.

Quantitative proteomics as a new piece of the systems biology puzzle

The definition of the role of each gene product in its cellular context is of outstanding importance in the post-genomics era. Recent technological innovations have driven research in proteomics from single protein characterization to global approaches, aiming to achieve a comprehensive qualitative and quantitative description of complex molecular mechanisms. In this review, we discuss the methodology of quantitative proteomics as it applies to the analysis of complex biological model systems. A special attention will be given to model systems that are suitable for functional genomic studies, where the potential of quantitative proteomics can be effectively demonstrated.

Yin Yang 1 extends the Myc-related transcription factors network in embryonic stem cells

The Yin Yang 1 (YY1) transcription factor is a master regulator of development, essential for early embryogenesis and adult tissues formation. YY1 is the mammalian orthologue of Pleiohomeotic, one of the transcription factors that binds Polycomb DNA response elements in Drosophila melanogaster and mediates Polycomb group proteins (PcG) recruitment to DNA. Despite several publications pointing at YY1 having a similar role in mammalians, others showed features of YY1 that are not compatible with PcG functions. Here, we show that, in mouse Embryonic Stem (ES) cells, YY1 has genome-wide PcG-independent activities while it is still stably associated with the INO80 chromatin-remodeling complex, as well as with novel RNA helicase activities. YY1 binds chromatin in close proximity of the transcription start site of highly expressed genes. Loss of YY1 functions preferentially led to a down-regulation of target genes expression, as well as to an up-regulation of several small non-coding RNAs, suggesting a role for YY1 in regulating small RNA biogenesis. Finally, we found that YY1 is a novel player of Myc-related transcription factors and that its coordinated binding at promoters potentiates gene expression, proposing YY1 as an active component of the Myc transcription network that links ES to cancer cells.

SILAC-based proteomic analysis to dissect the "histone modification signature" of human breast cancer cells

In living cells, the N-terminal tails of core histones, the proteinaceous component of nucleosomes, are subjected to a range of covalent post-translational modifications (PTMs), which have specific roles in modulating chromatin structure and function. A growing body of evidence suggests that deregulation of histone modification patterns, upstream or downstream of DNA methylation, is a critical event in cancer initiation and progression. However, a comprehensive description of how histone modifications, singly or in combination, is disrupted in transformed cells is missing; consequently the issue whether and how specific changes in histone PTMs patterns correlate to particular tumor features is still elusive. In the present study, we focused on human breast cancer and comprehensively analyzed PTMs on histone H3 and H4 from four cancer cell lines (MCF7, MDA-MB231, MDA-MB453 and T-47D), in comparison with normal epithelial breast cells. We performed high-resolution mass spectrometry analysis of histones, in combination with stable isotope labeling with amino acids in cell culture (SILAC), to quantitatively track the modification changes in cancer cells, as compared to their normal counterpart. Our investigation focuses on lysine acetylation and methylation on fourteen distinct sites in H3 and H4. We observed significant changes for several modifications in cancer cells: while in a few cases those modifications had been previously described as a hallmark of human tumors, we could identify novel modifications, whose abundance is significantly altered in breast cancer cells. Overall, these modifications may represent part of a “breast cancer-specific epigenetic signature”, with implications in the characterization of histone-related biomarkers. This work demonstrates that SILAC-based proteomics is a powerful tool to study qualitatively and quantitatively histone PTMs patterns, contributing significantly to the comprehension of epigenetic phenomena in cancer biology.

Cell Reprogramming Requires Silencing of a Core Subset of Polycomb Targets

Transcription factor (TF)–induced reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is associated with genome-wide changes in chromatin modifications. Polycomb-mediated histone H3 lysine-27 trimethylation (H3K27me3) has been proposed as a defining mark that distinguishes the somatic from the iPSC epigenome. Here, we dissected the functional role of H3K27me3 in TF–induced reprogramming through the inactivation of the H3K27 methylase EZH2 at the onset of reprogramming. Our results demonstrate that surprisingly the establishment of functional iPSC proceeds despite global loss of H3K27me3. iPSC lacking EZH2 efficiently silenced the somatic transcriptome and differentiated into tissues derived from the three germ layers. Remarkably, the genome-wide analysis of H3K27me3 in Ezh2 mutant iPSC cells revealed the retention of this mark on a highly selected group of Polycomb targets enriched for developmental regulators controlling the expression of lineage specific genes. Erasure of H3K27me3 from these targets led to a striking impairment in TF–induced reprogramming. These results indicate that PRC2-mediated H3K27 trimethylation is required on a highly selective core of Polycomb targets whose repression enables TF–dependent cell reprogramming.

The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components

Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational modifications, histone variants, and DNA methylation. Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. Here we describe a modified protocol for chromatin immunoprecipitation combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH, both enriched in silent domains. Chromatin proteomics expands the arsenal of tools for deciphering how all the distinct protein components act together to enforce a given region-specific chromatin status.

Methylation of CenH3 arginine 37 regulates kinetochore integrity and chromosome segregation

Centromeres of eukaryotic chromosomes mark the site for kinetochore formation and microtubule attachment and are essential for accurate chromosome segregation. Although centromere identity is defined by the presence of the histone H3 variant CenH3/centromere protein A (CENP-A), little is known about how epigenetic modifications on CenH3 might regulate kinetochore assembly and centromere function. Here we show that CENP-A from Saccharomyces cerevisiae, termed Cse4, is methylated on arginine 37 (R37) and that this methylation regulates the recruitment of kinetochore components to centromeric sequences. The absence of Cse4 R37 methylation caused a growth defect in cells lacking the centromere binding factor Cbf1 and synthetic lethality when combined with mutations in components of the Ctf19 linker complex that connects the inner kinetochore to microtubule-binding proteins. The cells showed a cell-cycle arrest in G2/M phase and defects in plasmid and chromosome segregation. Furthermore, the levels of Mtw1/MIND (Mtw1 including Nnf1-Nsl1-Dsn1) and Ctf19 components at the centromere, but not of Cse4 itself, were reduced in the absence of Cse4 R37 methylation, thus showing that this modification regulates the recruitment of linker components to the centromere. Altogether, our data identify a unique regulatory principle on centromeric chromatin by posttranslational modification of the amino terminus of CenH3.