mTert induction in p21-positive cells counteracts capillary rarefaction and pulmonary emphysema.

Lung diseases develop when telomeres shorten beyond a critical point. We constructed a mouse model in which the catalytic subunit of telomerase (mTert), or its catalytically inactive form (mTertCI), is expressed from the p21Cdkn1a locus. Expression of either TERT or TERTCI reduces global p21 levels in the lungs of aged mice, highlighting TERT non-canonical function. However, only TERT reduces accumulation of very short telomeres, oxidative damage, endothelial cell (ECs) senescence and senile emphysema in aged mice. Single-cell analysis of the lung reveals that p21 (and hence TERT) is expressed mainly in the capillary ECs. We report that a fraction of capillary ECs marked by CD34 and endowed with proliferative capacity declines drastically with age, and this is counteracted by TERT but not TERTCI. Consistently, only TERT counteracts decline of capillary density. Natural aging effects are confirmed using the experimental model of emphysema induced by VEGFR2 inhibition and chronic hypoxia. We conclude that catalytically active TERT prevents exhaustion of the putative CD34 + EC progenitors with age, thus protecting against capillary vessel loss and pulmonary emphysema.

Modeling Heterogeneity of Triple-Negative Breast Cancer Uncovers a Novel Combinatorial Treatment Overcoming Primary Drug Resistance.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype characterized by a remarkable molecular heterogeneity. Currently, there are no effective druggable targets and advanced preclinical models of the human disease. Here, a unique mouse model (MMTV-R26Met mice) of mammary tumors driven by a subtle increase in the expression of the wild-type MET receptor is generated. MMTV-R26Met mice develop spontaneous, exclusive TNBC tumors, recapitulating primary resistance to treatment of patients. Proteomic profiling of MMTV-R26Met tumors and machine learning approach show that the model faithfully recapitulates intertumoral heterogeneity of human TNBC. Further signaling network analysis highlights potential druggable targets, of which cotargeting of WEE1 and BCL-XL synergistically kills TNBC cells and efficiently induces tumor regression. Mechanistically, BCL-XL inhibition exacerbates the dependency of TNBC cells on WEE1 function, leading to Histone H3 and phosphoS33RPA32 upregulation, RRM2 downregulation, cell cycle perturbation, mitotic catastrophe, and apoptosis. This study introduces a unique, powerful mouse model for studying TNBC formation and evolution, its heterogeneity, and for identifying efficient therapeutic targets.

Author Correction: Involvement of G-quadruplex regions in mammalian replication origin activity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Involvement of G-quadruplex regions in mammalian replication origin activity.

Genome-wide studies of DNA replication origins revealed that origins preferentially associate with an Origin G-rich Repeated Element (OGRE), potentially forming G-quadruplexes (G4). Here, we functionally address their requirements for DNA replication initiation in a series of independent approaches. Deletion of the OGRE/G4 sequence strongly decreased the corresponding origin activity. Conversely, the insertion of an OGRE/G4 element created a new replication origin. This element also promoted replication of episomal EBV vectors lacking the viral origin, but not if the OGRE/G4 sequence was deleted. A potent G4 ligand, PhenDC3, stabilized G4s but did not alter the global origin activity. However, a set of new, G4-associated origins was created, whereas suppressed origins were largely G4-free. In vitro Xenopus laevis replication systems showed that OGRE/G4 sequences are involved in the activation of DNA replication, but not in the pre-replication complex formation. Altogether, these results converge to the functional importance of OGRE/G4 elements in DNA replication initiation.

Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication.

Among other targets, the protein lysine methyltransferase PR-Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4-20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. Here, we show that H4K20 mutation in mammalian cells, unlike in Drosophila, partially impairs S-phase progression and protects from DNA re-replication induced by stabilization of PR-Set7. Using Epstein-Barr virus-derived episomes, we further demonstrate that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4-20h is not sufficient to define an efficient origin per se, but rather serves as an enhancer for MCM2-7 helicase loading and replication activation at defined origins. Consistent with this, we find that Suv4-20h-mediated H4K20 tri-methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1-associated origins, which ensure proper replication timing of late-replicating heterochromatin domains. Altogether, these results reveal Suv4-20h-mediated H4K20 tri-methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes.

The gastrula transition reorganizes replication-origin selection in Caenorhabditis elegans.

Although some features underlying replication-origin activation in metazoan cells have been determined, little is known about their regulation during metazoan development. Using the nascent-strand purification method, here we identified replication origins throughout Caenorhabditis elegans embryonic development and found that the origin repertoire is thoroughly reorganized after gastrulation onset. During the pluripotent embryonic stages (pregastrula), potential cruciform structures and open chromatin are determining factors that establish replication origins. The observed enrichment of replication origins in transcription factor-binding sites and their presence in promoters of highly transcribed genes, particularly operons, suggest that transcriptional activity contributes to replication initiation before gastrulation. After the gastrula transition, when embryonic differentiation programs are set, new origins are selected at enhancers, close to CpG-island-like sequences, and at noncoding genes. Our findings suggest that origin selection coordinates replication initiation with transcriptional programs during metazoan development.

The chromatin environment shapes DNA replication origin organization and defines origin classes.

To unveil the still-elusive nature of metazoan replication origins, we identified them genome-wide and at unprecedented high-resolution in mouse ES cells. This allowed initiation sites (IS) and initiation zones (IZ) to be differentiated. We then characterized their genetic signatures and organization and integrated these data with 43 chromatin marks and factors. Our results reveal that replication origins can be grouped into three main classes with distinct organization, chromatin environment, and sequence motifs. Class 1 contains relatively isolated, low-efficiency origins that are poor in epigenetic marks and are enriched in an asymmetric AC repeat at the initiation site. Late origins are mainly found in this class. Class 2 origins are particularly rich in enhancer elements. Class 3 origins are the most efficient and are associated with open chromatin and polycomb protein-enriched regions. The presence of Origin G-rich Repeated elements (OGRE) potentially forming G-quadruplexes (G4) was confirmed at most origins. These coincide with nucleosome-depleted regions located upstream of the initiation sites, which are associated with a labile nucleosome containing H3K64ac. These data demonstrate that specific chromatin landscapes and combinations of specific signatures regulate origin localization. They explain the frequently observed links between DNA replication and transcription. They also emphasize the plasticity of metazoan replication origins and suggest that in multicellular eukaryotes, the combination of distinct genetic features and chromatin configurations act in synergy to define and adapt the origin profile.

Genome-scale identification of active DNA replication origins.

Understanding the nature of DNA replication origins in metazoan is quite challenging. In the absence of a genetic assay like in yeast, methods were devised based on the DNA structure, the visualization or quantification of the first nascent strands that are synthesized at origins, or on the localization of origin binding proteins. The purification and quantification of RNA-primed nascent DNA at origins during initiation of DNA synthesis is the most exhaustive and precise method to map active replication origins at present. We have upgraded this method to the level of reproducibility and enrichment required for genome-wide analyses by microarrays or deep sequencing. We detail here the protocol and the controls required at the different steps.

New insights into replication origin characteristics in metazoans.

We recently reported the identification and characterization of DNA replication origins (Oris) in metazoan cell lines. Here, we describe additional bioinformatic analyses showing that the previously identified GC-rich sequence elements form origin G-rich repeated elements (OGREs) that are present in 67% to 90% of the DNA replication origins from Drosophila to human cells, respectively. Our analyses also show that initiation of DNA synthesis takes place precisely at 160 bp (Drosophila) and 280 bp (mouse) from the OGRE. We also found that in most CpG islands, an OGRE is positioned in opposite orientation on each of the two DNA strands and detected two sites of initiation of DNA synthesis upstream or downstream of each OGRE. Conversely, Oris not associated with CpG islands have a single initiation site. OGRE density along chromosomes correlated with previously published replication timing data. Ori sequences centered on the OGRE are also predicted to have high intrinsic nucleosome occupancy. Finally, OGREs predict G-quadruplex structures at Oris that might be structural elements controlling the choice or activation of replication origins.

Conserved molecular interactions within the HBO1 acetyltransferase complexes regulate cell proliferation.

Acetyltransferase complexes of the MYST family with distinct substrate specificities and functions maintain a conserved association with different ING tumor suppressor proteins. ING complexes containing the HBO1 acetylase are a major source of histone H3 and H4 acetylation in vivo and play critical roles in gene regulation and DNA replication. Here, our molecular dissection of HBO1/ING complexes unravels the protein domains required for their assembly and function. Multiple PHD finger domains present in different subunits bind the histone H3 N-terminal tail with a distinct specificity toward lysine 4 methylation status. We show that natively regulated association of the ING4/5 PHD domain with HBO1-JADE determines the growth inhibitory function of the complex, linked to its tumor suppressor activity. Functional genomic analyses indicate that the p53 pathway is a main target of the complex, at least in part through direct transcription regulation at the initiation site of p21/CDKN1A. These results demonstrate the importance of ING association with MYST acetyltransferases in controlling cell proliferation, a regulated link that accounts for the reported tumor suppressor activities of these complexes.

Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features.

In metazoans, thousands of DNA replication origins (Oris) are activated at each cell cycle. Their genomic organization and their genetic nature remain elusive. Here, we characterized Oris by nascent strand (NS) purification and a genome-wide analysis in Drosophila and mouse cells. We show that in both species most CpG islands (CGI) contain Oris, although methylation is nearly absent in Drosophila, indicating that this epigenetic mark is not crucial for defining the activated origin. Initiation of DNA synthesis starts at the borders of CGI, resulting in a striking bimodal distribution of NS, suggestive of a dual initiation event. Oris contain a unique nucleotide skew around NS peaks, characterized by G/T and C/A overrepresentation at the 5' and 3' of Ori sites, respectively. Repeated GC-rich elements were detected, which are good predictors of Oris, suggesting that common sequence features are part of metazoan Oris. In the heterochromatic chromosome 4 of Drosophila, Oris correlated with HP1 binding sites. At the chromosome level, regions rich in Oris are early replicating, whereas Ori-poor regions are late replicating. The genome-wide analysis was coupled with a DNA combing analysis to unravel the organization of Oris. The results indicate that Oris are in a large excess, but their activation does not occur at random. They are organized in groups of site-specific but flexible origins that define replicons, where a single origin is activated in each replicon. This organization provides both site specificity and Ori firing flexibility in each replicon, allowing possible adaptation to environmental cues and cell fates.

MOF and histone H4 acetylation at lysine 16 are critical for DNA damage response and double-strand break repair.

The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Here we show that reduced levels of H4K16ac correlate with a defective DNA damage response (DDR) and double-strand break (DSB) repair to ionizing radiation (IR). The defect, however, is not due to altered expression of proteins involved in DDR. Abrogation of IR-induced DDR by MOF depletion is inhibited by blocking H4K16ac deacetylation. MOF was found to be associated with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a protein involved in nonhomologous end-joining (NHEJ) repair. ATM-dependent IR-induced phosphorylation of DNA-PKcs was also abrogated in MOF-depleted cells. Our data indicate that MOF depletion greatly decreased DNA double-strand break repair by both NHEJ and homologous recombination (HR). In addition, MOF activity was associated with general chromatin upon DNA damage and colocalized with the synaptonemal complex in male meiocytes. We propose that MOF, through H4K16ac (histone code), has a critical role at multiple stages in the cellular DNA damage response and DSB repair.

Programming DNA replication origins and chromosome organization.

During each cell cycle, thousands of DNA replication origins are activated in each cell of a metazoan organism. Although they appear site-specific, their usage and organization are rather plastic. Moreover, no strict sequence specificity has been observed in contrast to bacterial or Saccharomyces cerevisiae DNA replication origins. Epigenetic regulation linked to chromatin structure, chromosome organization, and transcription has been suggested to explain how DNA replication origins are selected and recognized by replication initiation factors. In this paper, we review these epigenetic features and discuss how, during the previous mitosis, chromosomal architecture might prepare DNA replication origins for a new cell cycle.

HBO1 HAT complexes target chromatin throughout gene coding regions via multiple PHD finger interactions with histone H3 tail.

The HBO1 HAT protein is the major source of histone H4 acetylation in vivo and has been shown to play critical roles in gene regulation and DNA replication. A distinctive characteristic of HBO1 HAT complexes is the presence of three PHD finger domains in two different subunits: tumor suppressor proteins ING4/5 and JADE1/2/3. Biochemical and functional analyses indicate that these domains interact with histone H3 N-terminal tail region, but with a different specificity toward its methylation status. Their combinatorial action is essential in regulating chromatin binding and substrate specificity of HBO1 complexes, as well as cell growth. Importantly, localization analyses on the human genome indicate that HBO1 complexes are enriched throughout the coding regions of genes, supporting a role in transcription elongation. These results underline the importance and versatility of PHD finger domains in regulating chromatin association and histone modification crosstalk within a single protein complex.

Molecular architecture of quartet MOZ/MORF histone acetyltransferase complexes.

The monocytic leukemia zinc finger protein MOZ and the related factor MORF form tetrameric complexes with ING5 (inhibitor of growth 5), EAF6 (Esa1-associated factor 6 ortholog), and the bromodomain-PHD finger protein BRPF1, -2, or -3. To gain new insights into the structure, function, and regulation of these complexes, we reconstituted them and performed various molecular analyses. We found that BRPF proteins bridge the association of MOZ and MORF with ING5 and EAF6. An N-terminal region of BRPF1 interacts with the acetyltransferases; the enhancer of polycomb (EPc) homology domain in the middle part binds to ING5 and EAF6. The association of BRPF1 with EAF6 is weak, but ING5 increases the affinity. These three proteins form a trimeric core that is conserved from Drosophila melanogaster to humans, although authentic orthologs of MOZ and MORF are absent in invertebrates. Deletion mapping studies revealed that the acetyltransferase domain of MOZ/MORF is sufficient for BRPF1 interaction. At the functional level, complex formation with BRPF1 and ING5 drastically stimulates the activity of the acetyltransferase domain in acetylation of nucleosomal histone H3 and free histones H3 and H4. An unstructured 18-residue region at the C-terminal end of the catalytic domain is required for BRPF1 interaction and may function as an "activation lid." Furthermore, BRPF1 enhances the transcriptional potential of MOZ and a leukemic MOZ-TIF2 fusion protein. These findings thus indicate that BRPF proteins play a key role in assembling and activating MOZ/MORF acetyltransferase complexes.

Role of Jade-1 in the histone acetyltransferase (HAT) HBO1 complex.

Regulation of global chromatin acetylation is important for chromatin remodeling. A small family of Jade proteins includes Jade-1L, Jade-2, and Jade-3, each bearing two mid-molecule tandem plant homology domain (PHD) zinc fingers. We previously demonstrated that the short isoform of Jade-1L protein, Jade-1, is associated with endogenous histone acetyltransferase (HAT) activity. It has been found that Jade-1L/2/3 proteins co-purify with a novel HAT complex, consisting of HBO1, ING4/5, and Eaf6. We investigated a role for Jade-1/1L in the HBO1 complex. When overexpressed individually, neither Jade-1/1L nor HBO1 affected histone acetylation. However, co-expression of Jade-1/1L and HBO1 increased acetylation of the bulk of endogenous histone H4 in epithelial cells in a synergistic manner, suggesting that Jade1/1L positively regulates HBO1 HAT activity. Conversely, small interfering RNA-mediated depletion of endogenous Jade resulted in reduced levels of H4 acetylation. Moreover, HBO1-mediated H4 acetylation activity was enhanced severalfold by the presence of Jade-1/1L in vitro. The removal of PHD fingers affected neither binding nor mutual Jade-1-HBO1 stabilization but completely abrogated the synergistic Jade-1/1L- and HBO1-mediated histone H4 acetylation in live cells and in vitro with reconstituted oligonucleosome substrates. Therefore, PHDs are necessary for Jade-1/1L-induced acetylation of nucleosomal histones by HBO1. In contrast to Jade-1/1L, the PHD zinc finger protein ING4/5 failed to synergize with HBO1 to promote histone acetylation. The physical interaction of ING4/5 with HBO1 occurred in the presence of Jade-1L or Jade-3 but not with the Jade-1 short isoform. In summary, this study demonstrates that Jade-1/1L are crucial co-factors for HBO1-mediated histone H4 acetylation.

ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression.

Dynamic regulation of diverse nuclear processes is intimately linked to covalent modifications of chromatin. Much attention has focused on methylation at lysine 4 of histone H3 (H3K4), owing to its association with euchromatic genomic regions. H3K4 can be mono-, di- or tri-methylated. Trimethylated H3K4 (H3K4me3) is preferentially detected at active genes, and is proposed to promote gene expression through recognition by transcription-activating effector molecules. Here we identify a novel class of methylated H3K4 effector domains--the PHD domains of the ING (for inhibitor of growth) family of tumour suppressor proteins. The ING PHD domains are specific and highly robust binding modules for H3K4me3 and H3K4me2. ING2, a native subunit of a repressive mSin3a-HDAC1 histone deacetylase complex, binds with high affinity to the trimethylated species. In response to DNA damage, recognition of H3K4me3 by the ING2 PHD domain stabilizes the mSin3a-HDAC1 complex at the promoters of proliferation genes. This pathway constitutes a new mechanism by which H3K4me3 functions in active gene repression. Furthermore, ING2 modulates cellular responses to genotoxic insults, and these functions are critically dependent on ING2 interaction with H3K4me3. Together, our findings establish a pivotal role for trimethylation of H3K4 in gene repression and, potentially, tumour suppressor mechanisms.

ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation.

Members of the ING family of tumor suppressors regulate cell cycle progression, apoptosis, and DNA repair as important cofactors of p53. ING1 and ING3 are stable components of the mSin3A HDAC and Tip60/NuA4 HAT complexes, respectively. We now report the purification of the three remaining human ING proteins. While ING2 is in an HDAC complex similar to ING1, ING4 associates with the HBO1 HAT required for normal progression through S phase and the majority of histone H4 acetylation in vivo. ING5 fractionates with two distinct complexes containing HBO1 or nucleosomal H3-specific MOZ/MORF HATs. These ING5 HAT complexes interact with the MCM helicase and are essential for DNA replication to occur during S phase. Our data also indicate that ING subunits are crucial for acetylation of chromatin substrates. Since INGs, HBO1, and MOZ/MORF contribute to oncogenic transformation, the multisubunit assemblies characterized here underscore the critical role of epigenetic regulation in cancer development.

A human protein complex homologous to the Drosophila MSL complex is responsible for the majority of histone H4 acetylation at lysine 16.

We describe a stable, multisubunit human histone acetyltransferase complex (hMSL) that contains homologs of the Drosophila dosage compensation proteins MOF, MSL1, MSL2, and MSL3. This complex shows strong specificity for histone H4 lysine 16 in chromatin in vitro, and RNA interference-mediated knockdown experiments reveal that it is responsible for the majority of H4 acetylation at lysine 16 in the cell. We also find that hMOF is a component of additional complexes, forming associations with host cell factor 1 and a protein distantly related to MSL1 (hMSL1v1). We find two versions of hMSL3 in the hMSL complex that differ by the presence of the chromodomain. Lastly, we find that reduction in the levels of hMSLs and acetylation of H4 at lysine 16 are correlated with reduced transcription of some genes and with a G(2)/M cell cycle arrest. This is of particular interest given the recent correlation of global loss of acetylation of lysine 16 in histone H4 with tumorigenesis.