DLK1/DIO3 locus upregulation by a ß-catenin-dependent enhancer drives cell proliferation and liver tumorigenesis.

The CTNNB1 gene, encoding ß-catenin, is frequently mutated in hepatocellular carcinoma (HCC, ∼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained ß-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (ApcΔhep) or Ctnnb1-exon 3 (ß-cateninΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/ß-catenin complexes in an open conformation upon sustained ß-catenin activation (DLK1-Wnt responsive element [WRE]) and increasing DLK1/DIO3 locus transcription in ß-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR-Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, ApcΔhep HB and ß-cateninΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during ß-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations.

Chromatin and DNA Dynamics in Mouse Models of Liver Cancers.

In recent years, important efforts have been made to understand how the expression of a specific gene repertoire correlates with chromatin accessibility, histone mark deposition, as well as with chromatin looping establishing connectivity with regulatory regions. The emergence of new techniques for genome-wide analyses and their progressive optimization to work on low amounts of material allows the scientific community to obtain an integrated view of transcriptional landscapes in physiology and disease. Here, we describe our own experience aiming at correlating the TCF-4/ß-catenin cistrome during liver tumorigenesis with chromatin remodeling, histone mark modifications, and long-distance DNA looping.

Global genome decompaction leads to stochastic activation of gene expression as a first step toward fate commitment in human hematopoietic cells.

When human cord blood-derived CD34+ cells are induced to differentiate, they undergo rapid and dynamic morphological and molecular transformations that are critical for fate commitment. In particular, the cells pass through a transitory phase known as "multilineage-primed" state. These cells are characterized by a mixed gene expression profile, different in each cell, with the coexpression of many genes characteristic for concurrent cell lineages. The aim of our study is to understand the mechanisms of the establishment and the exit from this transitory state. We investigated this issue using single-cell RNA sequencing and ATAC-seq. Two phases were detected. The first phase is a rapid and global chromatin decompaction that makes most of the gene promoters in the genome accessible for transcription. It results 24 h later in enhanced and pervasive transcription of the genome leading to the concomitant increase in the cell-to-cell variability of transcriptional profiles. The second phase is the exit from the multilineage-primed phase marked by a slow chromatin closure and a subsequent overall down-regulation of gene transcription. This process is selective and results in the emergence of coherent expression profiles corresponding to distinct cell subpopulations. The typical time scale of these events spans 48 to 72 h. These observations suggest that the nonspecificity of genome decompaction is the condition for the generation of a highly variable multilineage expression profile. The nonspecific phase is followed by specific regulatory actions that stabilize and maintain the activity of key genes, while the rest of the genome becomes repressed again by the chromatin recompaction. Thus, the initiation of differentiation is reminiscent of a constrained optimization process that associates the spontaneous generation of gene expression diversity to subsequent regulatory actions that maintain the activity of some genes, while the rest of the genome sinks back to the repressive closed chromatin state.

Identification of risk loci for primary aldosteronism in genome-wide association studies.

Primary aldosteronism affects up to 10% of hypertensive patients and is responsible for treatment resistance and increased cardiovascular risk. Here we perform a genome-wide association study in a discovery cohort of 562 cases and 950 controls and identify three main loci on chromosomes 1, 13 and X; associations on chromosome 1 and 13 are replicated in a second cohort and confirmed by a meta-analysis involving 1162 cases and 3296 controls. The association on chromosome 13 is specific to men and stronger in bilateral adrenal hyperplasia than aldosterone producing adenoma. Candidate genes located within the two loci, CASZ1 and RXFP2, are expressed in human and mouse adrenals in different cell clusters. Their overexpression in adrenocortical cells suppresses mineralocorticoid output under basal and stimulated conditions, without affecting cortisol biosynthesis. Our study identifies the first risk loci for primary aldosteronism and highlights new mechanisms for the development of aldosterone excess.

Exome-Wide Association Study Identifies FN3KRP and PGP as New Candidate Longevity Genes.

Despite enormous research efforts, the genetic component of longevity has remained largely elusive. The investigation of common variants, mainly located in intronic or regulatory regions, has yielded only little new information on the heritability of the phenotype. Here, we performed a chip-based exome-wide association study investigating 62 488 common and rare coding variants in 1248 German long-lived individuals, including 599 centenarians and 6941 younger controls (age < 60 years). In a single-variant analysis, we observed an exome-wide significant association between rs1046896 in the gene fructosamine-3-kinase-related-protein (FN3KRP) and longevity. Noteworthy, we found the longevity allele C of rs1046896 to be associated with an increased FN3KRP expression in whole blood; a database look-up confirmed this effect for various other human tissues. A gene-based analysis, in which potential cumulative effects of common and rare variants were considered, yielded the gene phosphoglycolate phosphatase (PGP) as another potential longevity gene, though no single variant in PGP reached the discovery p-value (1 × 10E-04). Furthermore, we validated the previously reported longevity locus cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1). Replication of our results in a French longevity cohort was only successful for rs1063192 in CDKN2B-AS1. In conclusion, we identified 2 new potential candidate longevity genes, FN3KRP and PGP which may influence the phenotype through their role in metabolic processes, that is, the reverse glycation of proteins (FN3KRP) and the control of glycerol-3-phosphate levels (PGP).

Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes.

Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. We investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BRDT, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.

Comparative methylome analysis of ICF patients identifies heterochromatin loci that require ZBTB24, CDCA7 and HELLS for their methylated state.

Alterations of DNA methylation landscapes and machinery are a hallmark of many human diseases. A prominent case is the ICF syndrome, a rare autosomal recessive immunological/neurological disorder diagnosed by the loss of DNA methylation at (peri)centromeric repeats and its associated chromosomal instability. It is caused by mutations in the de novo DNA methyltransferase DNMT3B in about half of the patients (ICF1). In the remainder, the striking identification of mutations in factors devoid of DNA methyltransferase activity, ZBTB24 (ICF2), CDCA7 (ICF3) or HELLS (ICF4), raised key questions about common or distinguishing DNA methylation alterations downstream of these mutations and hence, about the functional link between the four factors. Here, we established the first comparative methylation profiling in ICF patients with all four genotypes and we provide evidence that, despite unifying hypomethylation of pericentromeric repeats and a few common loci, methylation profiling clearly distinguished ICF1 from ICF2, 3 and 4 patients. Using available genomic and epigenomic annotations to characterize regions prone to loss of DNA methylation downstream of ICF mutations, we found that ZBTB24, CDCA7 and HELLS mutations affect CpG-poor regions with heterochromatin features. Among these, we identified clusters of coding and non-coding genes mostly expressed in a monoallelic manner and implicated in neuronal development, consistent with the clinical spectrum of these patients' subgroups. Hence, beyond providing blood-based biomarkers of dysfunction of ICF factors, our comparative study unveiled new players to consider at certain heterochromatin regions of the human genome.

Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells.

ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller-nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3' end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.

Histone H3 trimethylation at lysine 36 is associated with constitutive and facultative heterochromatin.

The mammalian genome contains numerous regions known as facultative heterochromatin, which contribute to transcriptional silencing during development and cell differentiation. We have analyzed the pattern of histone modifications associated with facultative heterochromatin within the mouse imprinted Snurf-Snrpn cluster, which is homologous to the human Prader-Willi syndrome genomic region. We show here that the maternally inherited Snurf-Snrpn 3-Mb region, which is silenced by a potent transcription repressive mechanism, is uniformly enriched in histone methylation marks usually found in constitutive heterochromatin, such as H4K20me3, H3K9me3, and H3K79me3. Strikingly, we found that trimethylated histone H3 at lysine 36 (H3K36me3), which was previously identified as a hallmark of actively transcribed regions, is deposited onto the silenced, maternally contributed 3-Mb imprinted region. We show that H3K36me3 deposition within this large heterochromatin domain does not correlate with transcription events, suggesting the existence of an alternative pathway for the deposition of this histone modification. In addition, we demonstrate that H3K36me3 is markedly enriched at the level of pericentromeric heterochromatin in mouse embryonic stem cells and fibroblasts. This result indicates that H3K36me3 is associated with both facultative and constitutive heterochromatin. Our data suggest that H3K36me3 function is not restricted to actively transcribed regions only and may contribute to the composition of heterochromatin, in combination with other histone modifications.

Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region.

X chromosome inactivation (XCI) is an essential epigenetic process which involves several non-coding RNAs (ncRNAs), including Xist, the master regulator of X-inactivation initiation. Xist is flanked in its 5' region by a large heterochromatic hotspot, which contains several transcription units including a gene of unknown function, Ftx (five prime to Xist). In this article, we describe the characterization and functional analysis of murine Ftx. We present evidence that Ftx produces a conserved functional long ncRNA, and additionally hosts microRNAs (miR) in its introns. Strikingly, Ftx partially escapes X-inactivation and is upregulated specifically in female ES cells at the onset of X-inactivation, an expression profile which closely follows that of Xist. We generated Ftx null ES cells to address the function of this gene. In these cells, only local changes in chromatin marks are detected within the hotspot, indicating that Ftx is not involved in the global maintenance of the heterochromatic structure of this region. The Ftx mutation, however, results in widespread alteration of transcript levels within the X-inactivation center (Xic) and particularly important decreases in Xist RNA levels, which were correlated with increased DNA methylation at the Xist CpG island. Altogether our results indicate that Ftx is a positive regulator of Xist and lead us to propose that Ftx is a novel ncRNA involved in XCI.

A role for non-coding Tsix transcription in partitioning chromatin domains within the mouse X-inactivation centre.

BACKGROUND: Delimiting distinct chromatin domains is essential for temporal and spatial regulation of gene expression. Within the X-inactivation centre region (Xic), the Xist locus, which triggers X-inactivation, is juxtaposed to a large domain of H3K27 trimethylation (H3K27me3). RESULTS: We describe here that developmentally regulated transcription of Tsix, a crucial non-coding antisense to Xist, is required to block the spreading of the H3K27me3 domain to the adjacent H3K4me2-rich Xist region. Analyses of a series of distinct Tsix mutations suggest that the underlying mechanism involves the RNA Polymerase II accumulating at the Tsix 3'-end. Furthermore, we report additional unexpected long-range effects of Tsix on the distal sub-region of the Xic, involved in Xic-Xic trans-interactions. CONCLUSION: These data point toward a role for transcription of non-coding RNAs as a developmental strategy for the establishment of functionally distinct domains within the mammalian genome.

Interactions between conserved domains within homodimers in the BIG1, BIG2, and GBF1 Arf guanine nucleotide exchange factors.

Guanine nucleotide exchange factors carrying a Sec7 domain (ArfGEFs) activate the small GTP-binding protein Arf, a major regulator of membrane remodeling and protein trafficking in eukaryotic cells. Only two of the seven subfamilies of ArfGEFs (GBF and BIG) are found in all eukaryotes. In addition to the Sec7 domain, which catalyzes GDP/GTP exchange on Arf, the GBF and BIG ArfGEFs have five common homology domains. Very little is known about the functions of these noncatalytic domains, but it is likely that they serve to integrate upstream signals that define the conditions of Arf activation. Here we describe interactions between two conserved domains upstream of the Sec7 domain (DCB and HUS) that determine the architecture of the N-terminal regions of the GBF and BIG ArfGEFs using a combination of biochemical, yeast two-hybrid, and cellular assays. Our data demonstrate a strong interaction between DCB domains within GBF1, BIG1, and BIG2 to maintain homodimers and an interaction between DCB and HUS domains within each homodimer. The DCB/HUS interaction is mediated by the HUS box, the most conserved motif in large ArfGEFs after the Sec7 domain. In support of the in vitro data, we show that both the DCB and the HUS domains are necessary for GBF1 dimerization in mammalian cells and that the DCB domain is essential for yeast viability. We propose that the dimeric DCB-HUS structural unit exists in all members of the GBF and BIG ArfGEF groups and in the related Mon2p family and probably serves an important regulatory role in Arf activation.

The Arf activator Gea2p and the P-type ATPase Drs2p interact at the Golgi in Saccharomyces cerevisiae.

Arf GTPases regulate both the morphological and protein sorting events that are essential for membrane trafficking. Guanine nucleotide exchange factors (GEFs) specific for Arf proteins determine when and where Arf GTPases will be activated in cells. The yeast Gea2p Arf GEF is a member of an evolutionarily conserved family of high molecular mass Arf GEFs that are peripherally associated with membranes. Nothing is known about how these proteins are localized to membranes, and few direct binding partners have been identified. In yeast, Gea2p has been implicated in trafficking through the Golgi apparatus and in maintaining Golgi structure. A major function of the Golgi apparatus is the packaging of cargo into secretory granules or vesicles. This process occurs through a series of membrane transformation events starting with fenestration of a saccular membrane, and subsequent remodeling of the fenestrated membrane into a mesh-like tubular network. Concentration of secretory cargo into nodes of the tubular network leads to enlargement of the nodes, which correspond to forming vesicles/granules, and thinning of the surrounding tubules. The tubules eventually break to release the secretory vesicles/granules into the cytoplasm. This process is highly conserved at the morphological level from yeast to mammalian cells. Drs2p, a multi-span transmembrane domain protein and putative aminophospholipid translocase, is required for the formation of a class of secretory granules/vesicles in yeast. Here we show that Drs2p interacts directly with Gea2p, both in vitro and in vivo. We mapped the domain of interaction of Drs2p to a 20-amino-acid region of the C-terminal cytoplasmic tail of the protein, adjacent to a region essential for Drs2p function. Mutations in Gea2p that abolish interaction with Drs2p are clustered in the C-terminal third of the Sec7 domain, and are important for Gea2p function. We characterize one such mutant that has a thermosensitive phenotype, and show that it has morphological defects along the secretory pathway in the formation of secretory granules/vesicles.

A novel Golgi membrane protein is a partner of the ARF exchange factors Gea1p and Gea2p.

The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics and protein trafficking. The interactions of large ARF GEFs with cellular membranes for localization and/or activation are likely to participate in regulated recruitment of ARF and effectors. However, these interactions remain largely unknown. Here we characterize Gmh1p, the first Golgi transmembrane-domain partner of any of the high-molecular-weight ARF-GEFs. Gmh1p is an evolutionarily conserved protein. We demonstrate molecular interaction between the yeast Gmh1p and the large ARF-GEFs Gea1p and Gea2p. This interaction involves a domain of Gea1p and Gea2p that is conserved in the eukaryotic orthologues of the Gea proteins. A single mutation in a conserved amino acid residue of this domain is sufficient to abrogate the interaction, whereas the overexpression of Gmh1p can compensate in vivo defects caused by mutations in this domain. We show that Gmh1p is an integral membrane protein that localizes to the early Golgi in yeast and in human HeLa cells and cycles through the ER. Hence, we propose that Gmh1p acts as a positive Golgi-membrane partner for Gea function. These results are of general interest given the evolutionary conservation of both ARF-GEFs and the Gmh proteins.