The CUE1 domain of the SNF2-like chromatin remodeler SMARCAD1 mediates its association with KRAB-associated protein 1 (KAP1) and KAP1 target genes.

Chromatin in embryonic stem cells (ESCs) differs markedly from that in somatic cells, with ESCs exhibiting a more open chromatin configuration. Accordingly, ATP-dependent chromatin remodeling complexes are important regulators of ESC homeostasis. Depletion of the remodeler SMARCAD1, an ATPase of the SNF2 family, has been shown to affect stem cell state, but the mechanistic explanation for this effect is unknown. Here, we set out to gain further insights into the function of SMARCAD1 in mouse ESCs. We identified KRAB-associated protein 1 (KAP1) as the stoichiometric binding partner of SMARCAD1 in ESCs. We found that this interaction occurs on chromatin and that SMARCAD1 binds to different classes of KAP1 target genes, including zinc finger protein (ZFP) and imprinted genes. We also found that the RING B-box coiled-coil (RBCC) domain in KAP1 and the proximal coupling of ubiquitin conjugation to ER degradation (CUE) domain in SMARCAD1 mediate their direct interaction. Of note, retention of SMARCAD1 in the nucleus depended on KAP1 in both mouse ESCs and human somatic cells. Mutations in the CUE1 domain of SMARCAD1 perturbed the binding to KAP1 in vitro and in vivo Accordingly, an intact CUE1 domain was required for tethering this remodeler to the nucleus. Moreover, mutation of the CUE1 domain compromised SMARCAD1 binding to KAP1 target genes. Taken together, our results reveal a mechanism that localizes SMARCAD1 to genomic sites through the interaction of SMARCAD1's CUE1 motif with KAP1.

Maintenance of silent chromatin through replication requires SWI/SNF-like chromatin remodeler SMARCAD1.

Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.

Global hypomethylation of the genome in XX embryonic stem cells.

Embryonic stem (ES) cells are important tools in the study of gene function and may also become important in cell therapy applications. Establishment of stable XX ES cell lines from mouse blastocysts is relatively problematic owing to frequent loss of one of the two X chromosomes. Here we show that DNA methylation is globally reduced in XX ES cell lines and that this is attributable to the presence of two active X chromosomes. Hypomethylation affects both repetitive and unique sequences, the latter including differentially methylated regions that regulate expression of parentally imprinted genes. Methylation of differentially methylated regions can be restored coincident with elimination of an X chromosome in early-passage parthenogenetic ES cells, suggesting that selection against loss of methylation may provide the basis for X-chromosome instability. Finally, we show that hypomethylation is associated with reduced levels of the de novo DNA methyltransferases Dnmt3a and Dnmt3b and that ectopic expression of these factors restores global methylation levels.

Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin.

Post-translational modifications of histone amino termini are an important regulatory mechanism that induce transitions in chromatin structure, thereby contributing to epigenetic gene control and the assembly of specialized chromosomal subdomains. Methylation of histone H3 at lysine 9 (H3-Lys9) by site-specific histone methyltransferases (Suv39h HMTases) marks constitutive heterochromatin. Here, we show that H3-Lys9 methylation also occurs in facultative heterochromatin of the inactive X chromosome (Xi) in female mammals. H3-Lys9 methylation is retained through mitosis, indicating that it might provide an epigenetic imprint for the maintenance of the inactive state. Disruption of the two mouse Suv39h HMTases abolishes H3-Lys9 methylation of constitutive heterochromatin but not that of the Xi. In addition, HP1 proteins, which normally associate with heterochromatin, do not accumulate with the Xi. These observations suggest the existence of an Suv39h-HP1-independent pathway regulating H3-Lys9 methylation of facultative heterochromatin.

The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse.

In vertebrates, SMARCAD1 participates in transcriptional regulation, heterochromatin maintenance, DNA repair, and replication. The molecular basis underlying its involvement in these processes is not well understood. We identified the RNA polymerase III general transcription factor TFIIIC as an interaction partner of native SMARCAD1 in mouse and human models using endogenous co-immunoprecipitations. TFIIIC has dual functionality, acting as a general transcription factor and as a genome organizer separating chromatin domains. We found that its partnership with SMARCAD1 is conserved across different mammalian cell types, from somatic to pluripotent cells. Using purified proteins, we confirmed that their interaction is direct. A gene expression analysis suggested that SMARCAD1 is dispensable for TFIIIC function as an RNA polymerase III transcription factor in mouse ESCs. The distribution of TFIIIC and SMARCAD1 in the ESC genome is distinct, and unlike in yeast, SMARCAD1 is not enriched at active tRNA genes. Further analysis of SMARCAD1-binding partners in pluripotent and differentiated mammalian cells reveals that SMARCAD1 associates with several factors that have key regulatory roles in chromatin organization, such as cohesin, laminB, and DDX5. Together, our work suggests for the first time that the SMARCAD1 enzyme participates in genome organization in mammalian nuclei through interactions with architectural proteins.

The Role of the m6A RNA Methyltransferase METTL16 in Gene Expression and SAM Homeostasis.

The RNA methylation of adenosine at the N6-position (m6A) has attracted significant attention because of its abundance and dynamic nature. It accounts for more than 80% of all RNA modifications present in bacteria and eukaryotes and regulates crucial aspects of RNA biology and gene expression in numerous biological processes. The majority of m6A found in mammals is deposited by a multicomponent complex formed between methyltransferase-like (METTL) proteins METTL3 and METTL14. In the last few years, the list of m6A writers has grown, resulting in an expansion of our understanding of the importance of m6A and the methylation machinery. The characterization of the less familiar family member METTL16 has uncovered a new function of the m6A methylation apparatus, namely the fine-tuning of the cellular levels of the major methyl donor S-adenosylmethionine (SAM). METTL16 achieves this by adjusting the levels of the enzyme that synthesizes SAM in direct response to fluctuations in the SAM availability. This review summarizes recent progress made in understanding how METTL16 can sense and relay metabolic information and considers the wider implications. A brief survey highlights similarities and differences between METTL16 and the better-known METTL3/14 complex, followed by a discussion of the target specificity, modes of action and potential roles of METTL16.

Neurite-like structures induced by mevalonate pathway blockade are due to the stability of cell adhesion foci and are enhanced by the presence of APP.

Epidemiological studies have shown an association between statin use and a decreased risk of dementia. However, the mechanism by which this beneficial effect is brought about is unclear. In the context of Alzheimer's disease, at least three possibilities have been studied; reduction in amyloid beta peptide (Abeta) production, the promotion of alpha-secretase cleavage and positive effects on neurite outgrowth. By investigating the effects of mevalonate pathway blockade on neurite outgrowth using real-time imaging, we found that rather than promote the production of neurite extensions, inhibition rapidly induced cell rounding. Crucially, neurite-like structures were generated through the persistence of cell-cell and cell-substrate adhesions and not through a mechanism of positive outgrowth. This effect can be strikingly enhanced by the over-expression of human amyloid precursor protein and is isoprenoid rather than cholesterol dependent.

Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation.

In many higher organisms, 5%-15% of histone H2A is ubiquitylated at lysine 119 (uH2A). The function of this modification and the factors involved in its establishment, however, are unknown. Here we demonstrate that uH2A occurs on the inactive X chromosome in female mammals and that this correlates with recruitment of Polycomb group (PcG) proteins belonging to Polycomb repressor complex 1 (PRC1). Based on our observations, we tested the role of the PRC1 protein Ring1B and its closely related homolog Ring1A in H2A ubiquitylation. Analysis of Ring1B null embryonic stem (ES) cells revealed extensive depletion of global uH2A levels. On the inactive X chromosome, uH2A was maintained in Ring1A or Ring1B null cells, but not in double knockout cells, demonstrating an overlapping function for these proteins in development. These observations link H2A ubiquitylation, X inactivation, and PRC1 PcG function, suggesting an unanticipated and novel mechanism for chromatin-mediated heritable gene silencing.

SMARCAD1 ATPase activity is required to silence endogenous retroviruses in embryonic stem cells.

Endogenous retroviruses (ERVs) can confer benefits to their host but present a threat to genome integrity if not regulated correctly. Here we identify the SWI/SNF-like remodeler SMARCAD1 as a key factor in the control of ERVs in embryonic stem cells. SMARCAD1 is enriched at ERV subfamilies class I and II, particularly at active intracisternal A-type particles (IAPs), where it preserves repressive histone methylation marks. Depletion of SMARCAD1 results in de-repression of IAPs and adjacent genes. Recruitment of SMARCAD1 to ERVs is dependent on KAP1, a central component of the silencing machinery. SMARCAD1 and KAP1 occupancy at ERVs is co-dependent and requires the ATPase function of SMARCAD1. Our findings uncover a role for the enzymatic activity of SMARCAD1 in cooperating with KAP1 to silence ERVs. This reveals ATP-dependent chromatin remodeling as an integral step in retrotransposon regulation in stem cells and advances our understanding of the mechanisms driving heterochromatin establishment.

Histone H3 lysine 9 methylation occurs rapidly at the onset of random X chromosome inactivation.

In female mammals, a single X chromosome is stably and heritably silenced early in embryogenesis. The inactive X is characterized by asynchronous DNA replication and epigenetic chromatin modifications, including DNA methylation, histone H3/H4 hypoacetylation, and incorporation of a variant histone macroH2A. X inactivation is initiated by a cis-acting RNA molecule, the X-inactive specific transcript (Xist), which coats the chromosome. However, the mechanism by which Xist induces chromosome silencing is poorly understood. An important approach towards answering this question has been to determine the temporal order of epigenetic chromatin modifications in an in vitro model system, differentiating XX embryonic stem (ES) cells, and thereby to identify candidate targets for Xist RNA. To date, these studies have demonstrated that, following accumulation of Xist RNA, the transition to late replication of the X chromosome is the earliest detectable event. H4 hypoacetylation, macroH2A1.2 incorporation, and DNA methylation all occur subsequently. Recently, it has been shown that chromatin of the inactive X is also characterized by methylation of histone H3 at lysine 9 (H3-K9). Here we show that H3-K9 methylation is a very early event in the process of X inactivation, which closely parallels the onset of Xist RNA accumulation.

Keeping chromatin quiet: how nucleosome remodeling restores heterochromatin after replication.

Disruption of chromatin organization during replication poses a major challenge to the maintenance and integrity of genome organization. It creates the need to accurately reconstruct the chromatin landscape following DNA duplication but there is little mechanistic understanding of how chromatin based modifications are restored on newly synthesized DNA. ATP-dependent chromatin remodeling activities serve multiple roles during replication and recent work underscores their requirement in the maintenance of proper chromatin organization. A new component of chromatin replication, the SWI/SNF-like chromatin remodeler SMARCAD1, acts at replication sites to facilitate deacetylation of newly assembled histones. Deacetylation is a pre-requisite for the restoration of epigenetic signatures in heterochromatin regions following replication. In this way, SMARCAD1, in concert with histone modifying activities and transcriptional repressors, reinforces epigenetic instructions to ensure that silenced loci are correctly perpetuated in each replication cycle. The emerging concept is that remodeling of nucleosomes is an early event imperative to promote the re-establishment of histone modifications following DNA replication.

Xist expression and macroH2A1.2 localisation in mouse primordial and pluripotent embryonic germ cells.

The molecular mechanism underlying X chromosome inactivation in female mammals involves the non-coding RNAs Xist and its antisense partner Tsix. Prior to X inactivation, these RNAs are transcribed in an unstable form from all X chromosomes, both in the early embryo and in undifferentiated embryonic stem (ES) cells. Upon differentiation, the expression of these unstable transcripts from all alleles is silenced, and Xist RNA becomes stabilised specifically on the inactivating X chromosome. This pattern of expression is then maintained throughout subsequent somatic cell divisions. Once established, the inactive state of the X chromosome is remarkably stable, the only natural case of reactivation occurring in XX primordial germ cells (PGCs) when they enter the genital ridge. To gain insight into the X reactivation process, we have analysed Xist gene expression using RNA FISH in PGCs and also in PGC-derived embryonic germ (EG) cells. XX EG cells were shown to express unstable Xist/Tsix from both X chromosomes. In contrast, no unstable Xist/Tsix transcripts were detected in XX PGCs at any stage. Instead, a proportion of XX PGCs isolated from the genital ridge between 11.5 and 13.5 dpc (the period during which X chromosome reactivation occurs) showed an accumulation of stable Xist RNA on one X. The number of these cells decreased progressively and was nearly extinguished by 13.5 dpc. As a late marker for the inactive state, we analysed localisation of the histone H2A variant macroH2A1.2. Although macroH2A1.2 expression was observed in PGCs, no significant localisation to the inactive X was detected at any stage. We discuss these results in the context of understanding X chromosome reactivation.