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The human forebrain has expanded in size and complexity compared to that of chimpanzee despite limited changes in protein-coding genes, suggesting that gene regulation is an important driver of brain evolution. Here we identify a KRAB-ZFP transcription factor, ZNF558, that is expressed in human but not chimpanzee forebrain neural progenitor cells. ZNF558 evolved as a suppressor of LINE-1 transposons but has been co-opted to regulate the mitophagy gene SPATA18, supporting a link between mitochondrial homeostasis and cortical expansion. The unusual on-off switch for ZNF558 expression resides in a downstream variable number tandem repeat (VNTR) that is contracted in humans relative to chimpanzee. Our data reveal the brain-specific co-option of a transposon-controlling KRAB-ZFP and how a human-specific regulatory network is established by a cis-acting structural genome variation. This represents a previously undescribed genetic mechanism in the evolution of the human brain.

The human forebrain has expanded in size and complexity compared to chimpanzees despite limited changes in protein-coding genes, suggesting that gene expression regulation is an important driver of brain evolution. Here, we identify a KRAB-ZFP transcription factor, ZNF558, that is expressed in human but not chimpanzee forebrain neural progenitor cells. ZNF558 evolved as a suppressor of LINE-1 transposons but has been co-opted to regulate a single target, the mitophagy gene SPATA18. ZNF558 plays a role in mitochondrial homeostasis, and loss-of-function experiments in cerebral organoids suggests that ZNF558 influences developmental timing during early human brain development. Expression of ZNF558 is controlled by the size of a variable number tandem repeat that is longer in chimpanzees compared to humans, and variable in the human population. Thus, this work provides mechanistic insight into how a cis-acting structural variation establishes a regulatory network that affects human brain evolution.

International audience; During cell division, maintenance of chromatin features from the parental genome requires their proper establishment on its newly synthetized copy. The loss of epigenetic marks within heterochromatin, typically enriched in repetitive elements, endangers genome stability and permits chromosomal rearrangements via recombination. However, how histone modifications associated with heterochromatin are maintained across mitosis remains poorly understood. KAP1 is known to act as a scaffold for a repressor complex that mediates local heterochromatin formation, and was previously demonstrated to play an important role during DNA repair. Accordingly, we investigated a putative role for this protein in the replication of heterochromatic regions. We first found that KAP1 associates with several DNA replication factors including PCNA, MCM3 and MCM6. We then observed that these interactions are promoted by KAP1 phosphorylation on serine 473 during S phase. Finally, we could demonstrate that KAP1 forms a complex with PCNA and the histone-lysine methyltransferase Suv39h1 to reinstate heterochromatin after DNA replication.

SETDB1 (SET Domain Bifurcated histone lysine methyltransferase 1) is a key lysine methyltransferase (KMT) required in embryonic stem cells (ESCs), where it silences transposable elements and DNA repeats via histone H3 lysine 9 tri-methylation (H3K9me3), independently of DNA methylation. The H3K9 methylation reader M-Phase Phosphoprotein 8 (MPP8) is highly expressed in ESCs and germline cells. Although evidence of a cooperation between H3K9 KMTs and MPP8 in committed cells has emerged, the interplay between H3K9 methylation writers and MPP8 in ESCs remains elusive. Here, we show that MPP8 interacts physically and functionally with SETDB1 in ESCs. Indeed, combining biochemical, transcriptomic and genomic analyses, we found that MPP8 and SETDB1 co-regulate a significant number of common genomic targets, especially the DNA satellite repeats. Together, our data point to a model in which the silencing of a class of repeated sequences in ESCs involves the cooperation between the H3K9 methylation writer SETDB1 and its reader MPP8. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.

The methylation of histone Lys residues by Lys methyltransferases (KMTs) regulates chromatin organization and either activates or represses gene expression, depending on the residue that is targeted. KMTs are emerging as key components in several cellular processes, and their deregulation is often associated with pathogenesis. Here, we review the current knowledge on the main KMTs that are associated with gene silencing: namely, those responsible for methylating histone H3 Lys 9 (H3K9), H3K27 and H4K20. We discuss their biochemical properties and the various mechanisms by which they are targeted to the chromatin and regulate gene expression, as well as new data on the interplay between them and other chromatin modifiers.

BCR ABL; International audience; BCR-ABL induces an intrinsic genetic instability in chronic myeloid leukemia (CML). The protein breast cancer 1, early onset (BRCA1)-associated protein 1 (BAP1) is a deubiquitinase interacting with the DNA repair regulator BRCA1 and is frequently inactivated in many cancers. Here, we report that BAP1 mRNA and protein levels are downregulated in a BCR-ABL1-expressing hematopoietic cell line (UT-7/11). A decrease of BAP1 transcripts is also observed in newly diagnosed CML patients. Moreover, BAP1 protein levels are low or undetectable in CD34(+) cells from CML patients at diagnosis as compared with CD34(+) cells from normal donors. In addition, BRCA1 protein level is reduced in BCR-ABL1-expressing UT-7/11 cells. Finally, the enforced expression of BAP1 is associated with BRCA1 protein deubiquitination and restoration. These results demonstrate BAP1 as a major link with the BCR-ABL-induced downregulation of BRCA1 in CML.

Significance: Methylation of histone H3 on lysine 9 and 27 (H3K9 and H3K27) are two epigenetic modifications that have been linked to several crucial biological processes, among which are transcriptional silencing and cell differentiation. Recent Advances: Deposition of these marks is catalyzed by H3K9 lysine methyltransferases (KMTs) and polycomb repressive complex 2, respectively. Increasing evidence is emerging in favor of a functional crosstalk between these two major KMT families. Critical Issues: Here, we review the current knowledge on the mechanisms of action and function of these enzymes, with particular emphasis on their interplay in the regulation of chromatin states and biological processes. We outline their crucial roles played in tissue homeostasis, by controlling the fate of embryonic and tissue-specific stem cells, highlighting how their deregulation is often linked to the emergence of a number of malignancies and neurological disorders. Future Directions: Histone methyltransferases are starting to be tested as drug targets. A new generation of highly selective chemical inhibitors is starting to emerge. These hold great promise for a rapid translation of targeting epigenetic drugs into clinical practice for a number of aggressive cancers and neurological disorders. Antioxid. Redox Signal. 22, 1365–1381.

Heterochromatic domains are enriched with repressive histone marks, including histone H3 lysine 9 methylation, written by lysine methyltransferases (KMTs). The pre-replication complex protein, origin recognition complex-associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells. Its role in heterochromatin organization remained elusive. ORCA recognizes methylated H3K9 marks and interacts with repressive KMTs, including G9a/GLP and Suv39H1 in a chromatin context-dependent manner. Single-molecule pull-down assays demonstrate that ORCA-ORC (Origin Recognition Complex) and multiple H3K9 KMTs exist in a single complex and that ORCA stabilizes H3K9 KMT complex. Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites. Changes in heterochromatin structure due to loss of ORCA affect replication timing, preferentially at the late-replicating regions. We demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure. DOI: http://dx.doi.org/10.7554/eLife.06496.001, eLife digest The genetic material inside cells is contained within molecules of DNA. In animals and other eukaryotes, the DNA is tightly wrapped around proteins called histones to form a compact structure known as chromatin. There are two forms of chromatin: loosely packed chromatin tends to contain genes that are highly active in cells, while tightly packed chromatin—called heterochromatin—tends to contain less-active genes. How tightly DNA is packed in chromatin can be changed by adding small molecules known as methyl tags to individual histone proteins. Enzymes called KMTs are responsible for attaching these methyl tags to a specific site on the histones. Before a cell divides, it duplicates its DNA and these methyl tags, so that they can be passed onto the newly formed cells. This enables the new cells to ‘remember’ which genes were inactive or active in the original cell. A protein known as ORCA associates with heterochromatin, but it is not clear what role it plays in controlling the structure of chromatin. Giri et al. studied ORCA and the KMTs in human cells. The experiments show that ORCA recognizes the methyl tags and binds to the KMTs in regions of heterochromatin, but not in regions where the DNA is more loosely packed. Next, Giri et al. used a technique called single-molecule pull-down, which is able to identify individual proteins within a group. These experiments showed that several KMT enzymes can bind to a single ORCA protein at the same time. ORCA stabilizes the binding of KMTs to chromatin, which enables the KMTs to modify the histones within it. Cells lacking ORCA had fewer methyl tags on their histones, which altered the structure of the chromatin. This also affected the timing with which DNA copying takes place in cells before the cell divides. Giri et al.'s findings suggest that ORCA acts as a scaffold for the KMTs and that its activity at specific sites on chromatin is important for the organization of heterochromatin. The next step is to identify the exact regions in the genome where the timing of DNA copying is regulated by ORCA. DOI: http://dx.doi.org/10.7554/eLife.06496.002

Myogenic terminal differentiation is a well-orchestrated process starting with permanent cell cycle exit followed by muscle-specific genetic program activation. Individual SWI/SNF components have been involved in muscle differentiation. Here, we show that the master myogenic differentiation factor MyoD interacts with more than one SWI/SNF subunit, including the catalytic subunit BRG1, BAF53a and the tumor suppressor BAF47/INI1. Downregulation of each of these SWI/SNF subunits inhibits skeletal muscle terminal differentiation but, interestingly, at different differentiation steps and extents. BAF53a downregulation inhibits myotube formation but not the expression of early muscle-specific genes. BRG1 or BAF47 downregulation disrupt both proliferation and differentiation genetic programs expression. Interestingly, BRG1 and BAF47 are part of the SWI/SNF remodeling complex as well as the N-CoR-1 repressor complex in proliferating myoblasts. However, our data show that, upon myogenic differentiation, BAF47 shifts in favor of N-CoR-1 complex. Finally, BRG1 and BAF47 are well-known tumor suppressors but, strikingly, only BAF47 seems essential in the myoblasts irreversible cell cycle exit. Together, our data unravel differential roles for SWI/SNF subunits in muscle differentiation, with BAF47 playing a dual role both in the permanent cell cycle exit and in the regulation of muscle-specific genes.

G9a/GLP and Polycomb Repressive Complex 2 (PRC2) are two major epigenetic silencing machineries, which in particular methylate histone H3 on lysines 9 and 27 (H3K9 and H3K27), respectively. Although evidence of a crosstalk between H3K9 and H3K27 methylations has started to emerge, their actual interplay remains elusive. Here, we show that PRC2 and G9a/GLP interact physically and functionally. Moreover, combining different genome-wide approaches, we demonstrate that Ezh2 and G9a/GLP share an important number of common genomic targets, encoding developmental and neuronal regulators. Furthermore, we show that G9a enzymatic activity modulates PRC2 genomic recruitment to a subset of its target genes. Taken together, our findings demonstrate an unanticipated interplay between two main histone lysine methylation mechanisms, which cooperate to maintain silencing of a subset of developmental genes.

International audience; Symmetrical dimethylation on arginine-3 of histone H4 (H4R3me2s) has been reported to occur at several repressed genes, but its specific regulation and genomic distribution remained unclear. Here, we show that the type-II protein arginine methyltransferase PRMT5 controls H4R3me2s in mouse embryonic fibroblasts (MEFs). In these differentiated cells, we find that the genome-wide pattern of H4R3me2s is highly similar to that in embryonic stem cells. In both the cell types, H4R3me2s peaks are detected predominantly at G + C-rich regions. Promoters are consistently marked by H4R3me2s, independently of transcriptional activity. Remarkably, H4R3me2s is mono-allelic at imprinting control regions (ICRs), at which it marks the same parental allele as H3K9me3, H4K20me3 and DNA methylation. These repressive chromatin modifications are regulated independently, however, since PRMT5-depletion in MEFs resulted in loss of H4R3me2s, without affecting H3K9me3, H4K20me3 or DNA methylation. Conversely, depletion of ESET (KMT1E) or SUV420H1/H2 (KMT5B/C) affected H3K9me3 and H4K20me3, respectively, without altering H4R3me2s at ICRs. Combined, our data indicate that PRMT5-mediated H4R3me2s uniquely marks the mammalian genome, mostly at G + C-rich regions, and independently from transcriptional activity or chromatin repression. Furthermore, comparative bioinformatics analyses suggest a putative role of PRMT5-mediated H4R3me2s in chromatin configuration in the nucleus.

The H19 gene controls the expression of several genes within the Imprinted Gene Network (IGN), involved in growth control of the embryo. However, the underlying mechanisms of this control remain elusive. Here, we identified the methyl-CpG-binding domain protein 1 MBD1 as a physical and functional partner of the H19 long noncoding RNA (lncRNA). The H19 lncRNA-MBD1 complex is required for the control of five genes of the IGN. For three of these genes--Igf2 (insulin-like growth factor 2), Slc38a4 (solute carrier family 38 member 4), and Peg1 (paternally expressed gene 1)--both MBD1 and H3K9me3 binding were detected on their differentially methylated regions. The H19 lncRNA-MBD1 complex, through its interaction with histone lysine methyltransferases, therefore acts by bringing repressive histone marks on the differentially methylated regions of these three direct targets of the H19 gene. Our data suggest that, besides the differential DNA methylation found on the differentially methylated regions of imprinted genes, an additional fine tuning of the expressed allele is achieved by a modulation of the H3K9me3 marks, mediated by the association of the H19 lncRNA with chromatin-modifying complexes, such as MBD1. This results in a precise control of the level of expression of growth factors in the embryo.

The histone 3 lysine 9 methyltransferase Setdb1 is essential for both stem cell pluripotency and terminal differentiation of different cell types. To shed light on the roles of Setdb1 in these mutually exclusive processes, we used mouse skeletal myoblasts as a model of terminal differentiation. Ex vivo studies on isolated single myofibres showed that Setdb1 is required for adult muscle stem cells expansion following activation. In vitro studies in skeletal myoblasts confirmed that Setdb1 suppresses terminal differentiation. Genomic binding analyses showed a release of Setdb1 from selected target genes upon myoblast terminal differentiation, concomitant to a nuclear export of Setdb1 to the cytoplasm. Both genomic release and cytoplasmic Setdb1 relocalisation during differentiation were dependent on canonical Wnt signalling. Transcriptomic assays in myoblasts unravelled a significant overlap between Setdb1 and Wnt3a regulated genetic programmes. Together, our findings revealed Wnt-dependent subcellular relocalisation of Setdb1 as a novel mechanism regulating Setdb1 functions and myogenesis.

Young transposable elements and their protein controllers team up to regulate the differentiation and function of human neurons., In the first days of embryogenesis, transposable element–embedded regulatory sequences (TEeRS) are silenced by Kruppel-associated box (KRAB) zinc finger proteins (KZFPs). Many TEeRS are subsequently co-opted in transcription networks, but how KZFPs influence this process is largely unknown. We identify ZNF417 and ZNF587 as primate-specific KZFPs repressing HERVK (human endogenous retrovirus K) and SVA (SINE-VNTR-Alu) integrants in human embryonic stem cells (ESCs). Expressed in specific regions of the human developing and adult brain, ZNF417/587 keep controlling TEeRS in ESC-derived neurons and brain organoids, secondarily influencing the differentiation and neurotransmission profile of neurons and preventing the induction of neurotoxic retroviral proteins and an interferon-like response. Thus, evolutionarily recent KZFPs and their TE targets partner up to influence human neuronal differentiation and physiology.

Expansion of transposable elements (TEs) coincides with evolutionary shifts in gene expression. TEs frequently harbor binding sites for transcriptional regulators, thus enabling coordinated genome-wide activation of species- and context-specific gene expression programs, but such regulation must be balanced against their genotoxic potential. Here, we show that Krüppel-associated box (KRAB)-containing zinc finger proteins (KZFPs) control the timely and pleiotropic activation of TE-derived transcriptional cis regulators during early embryogenesis. Evolutionarily recent SVA, HERVK, and HERVH TE subgroups contribute significantly to chromatin opening during human embryonic genome activation and are KLF-stimulated enhancers in naive human embryonic stem cells (hESCs). KZFPs of corresponding evolutionary ages are simultaneously induced and repress the transcriptional activity of these TEs. Finally, the same KZFP-controlled TE-based enhancers later serve as developmental and tissue-specific enhancers. Thus, by controlling the transcriptional impact of TEs during embryogenesis, KZFPs facilitate their genome-wide incorporation into transcriptional networks, thereby contributing to human genome regulation. Transposable elements (TEs) are key to the evolutionary turnover of regulatory sequences but potentially toxic to the host. Trono and colleagues demonstrate that KRAB zinc-finger proteins tame the activity of TEs during human early embryogenesis, thus allowing for their genome-wide incorporation into species-specific transcriptional networks., National Institutes of Health (Grant R37HD045022), National Institutes of Health (Grant R01-NS088538)

Germ cells are essential to pass DNA from one generation to the next. In human reproduction, germ cell development begins with the specification of primordial germ cells (PGCs) and a failure to specify PGCs leads to human infertility. Recent studies have revealed that the transcription factor network required for PGC specification has diverged in mammals, and this has a significant impact on our understanding of human reproduction. Here, we reveal that the Hominidae-specific Transposable Elements (TEs) LTR5Hs, may serve as TEENhancers (TE Embedded eNhancers) to facilitate PGC specification. LTR5Hs TEENhancers become transcriptionally active during PGC specification both in vivo and in vitro with epigenetic reprogramming leading to increased chromatin accessibility, localized DNA demethylation, enrichment of H3K27ac, and occupation of key hPGC transcription factors. Inactivation of LTR5Hs TEENhancers with KRAB mediated CRISPRi has a significant impact on germ cell specification. In summary, our data reveals the essential role of Hominidae-specific LTR5Hs TEENhancers in human germ cell development., The transcription factor network required for primordial germ cell (PGC) specification is known to diverge in mammals. Here the authors show that hominidae-specific transposable element (TE) LTR5Hs becomes transcriptionally active during PGC specification, and LTR5Hs inactivation abrogates human PGC specification