DNMT3B PWWP mutations cause hypermethylation of heterochromatin.

The correct establishment of DNA methylation patterns is vital for mammalian development and is achieved by the de novo DNA methyltransferases DNMT3A and DNMT3B. DNMT3B localises to H3K36me3 at actively transcribing gene bodies via its PWWP domain. It also functions at heterochromatin through an unknown recruitment mechanism. Here, we find that knockout of DNMT3B causes loss of methylation predominantly at H3K9me3-marked heterochromatin and that DNMT3B PWWP domain mutations or deletion result in striking increases of methylation in H3K9me3-marked heterochromatin. Removal of the N-terminal region of DNMT3B affects its ability to methylate H3K9me3-marked regions. This region of DNMT3B directly interacts with HP1α and facilitates the bridging of DNMT3B with H3K9me3-marked nucleosomes in vitro. Our results suggest that DNMT3B is recruited to H3K9me3-marked heterochromatin in a PWWP-independent manner that is facilitated by the protein's N-terminal region through an interaction with a key heterochromatin protein. More generally, we suggest that DNMT3B plays a role in DNA methylation homeostasis at heterochromatin, a process which is disrupted in cancer, aging and Immunodeficiency, Centromeric Instability and Facial Anomalies (ICF) syndrome.

Separable roles for RNAi in regulation of transposable elements and viability in the fission yeast Schizosaccharomyces japonicus.

RNA interference (RNAi) is a conserved mechanism of small RNA-mediated genome regulation commonly involved in suppression of transposable elements (TEs) through both post-transcriptional silencing, and transcriptional repression via heterochromatin assembly. The fission yeast Schizosaccharomyces pombe has been extensively utilised as a model for studying RNAi pathways. However, this species is somewhat atypical in that TEs are not major targets of RNAi, and instead small RNAs correspond primarily to non-coding pericentromeric repeat sequences, reflecting a specialised role for the pathway in promoting heterochromatin assembly in these regions. In contrast, in the related fission yeast Schizosaccharomyces japonicus, sequenced small RNAs correspond primarily to TEs. This suggests there may be fundamental differences in the operation of RNAi pathways in these two related species. To investigate these differences, we probed RNAi function in S. japonicus. Unexpectedly, and in contrast to S. pombe, we found that RNAi is essential in this species. Moreover, viability of RNAi mutants can be rescued by mutations implicated in enhancing RNAi-independent heterochromatin propagation. These rescued strains retain heterochromatic marks on TE sequences, but exhibit derepression of TEs at the post-transcriptional level. Our findings indicate that S. japonicus retains the ancestral role of RNAi in facilitating suppression of TEs via both post-transcriptional silencing and heterochromatin assembly, with specifically the heterochromatin pathway being essential for viability, likely due to a function in genome maintenance. The specialised role of RNAi in heterochromatin assembly in S. pombe appears to be a derived state that emerged after the divergence of S. japonicus.

Mkt1 is required for RNAi-mediated silencing and establishment of heterochromatin in fission yeast.

Constitutive domains of repressive heterochromatin are maintained within the fission yeast genome through self-reinforcing mechanisms involving histone methylation and small RNAs. Non-coding RNAs generated from heterochromatic regions are processed into small RNAs by the RNA interference pathway, and are subject to silencing through both transcriptional and post-transcriptional mechanisms. While the pathways involved in maintenance of the repressive heterochromatin state are reasonably well understood, less is known about the requirements for its establishment. Here, we describe a novel role for the post-transcriptional regulatory factor Mkt1 in establishment of heterochromatin at pericentromeres in fission yeast. Loss of Mkt1 does not affect maintenance of existing heterochromatin, but does affect its recovery following depletion, as well as de novo establishment of heterochromatin on a mini-chromosome. Pathway dissection revealed that Mkt1 is required for RNAi-mediated post-transcriptional silencing, downstream of small RNA production. Mkt1 physically associates with pericentromeric transcripts, and is additionally required for maintenance of silencing and heterochromatin at centromeres when transcriptional silencing is impaired. Our findings provide new insight into the mechanism of RNAi-mediated post-transcriptional silencing in fission yeast, and unveil an important role for post-transcriptional silencing in establishment of heterochromatin that is dispensable when full transcriptional silencing is imposed.

Structural analysis of Stc1 provides insights into the coupling of RNAi and chromatin modification.

Noncoding RNAs can modulate gene expression by directing modifications to histones that alter chromatin structure. In fission yeast, siRNAs produced via the RNAi pathway direct modifications associated with heterochromatin formation. siRNAs associate with the RNAi effector protein Argonaute 1 (Ago1), targeting the Ago1-containing RNA-induced transcriptional silencing (RITS) complex to homologous nascent transcripts. This promotes recruitment of the Clr4 complex (CLRC), which mediates methylation of histone H3 on lysine 9 (H3K9me) in cognate chromatin. A key question is how the RNAi and chromatin modification machineries are connected. Stc1 is a small protein recently shown to associate with both Ago1 and CLRC and to play a pivotal role in mediating the RNAi-dependent recruitment of CLRC to chromatin. To understand its mode of action, we have performed a detailed structural and functional analysis of the Stc1 protein. Our analyses reveal that the conserved N-terminal region of Stc1 represents an unusual tandem zinc finger domain, with similarities to common LIM domains but distinguished by a lack of preferred relative orientation of the two zinc fingers. We demonstrate that this tandem zinc finger domain is involved in binding Ago1, whereas the nonconserved C-terminal region mediates association with CLRC. These findings elucidate the molecular basis for the coupling of RNAi to chromatin modification in fission yeast.

The Gcm/Glide molecular and cellular pathway: new actors and new lineages.

In Drosophila, the transcription factor Gcm/Glide plays a key role in cell fate determination and cellular differentiation. In light of its crucial biological impact, major efforts have been put for analyzing its properties as master regulator, from both structural and functional points of view. However, the lack of efficient antibodies specific to the Gcm/Glide protein precluded thorough analyses of its regulation and activity in vivo. In order to relieve such restraints, we designed an epitope-tagging approach to "FLAG"-recognize and analyze the functional protein both in vitro (exogenous Gcm/Glide) and in vivo (endogenous protein). We here (i) reveal a tight interconnection between the small RNA and the Gcm/Glide pathways. AGO1 and miR-1 are Gcm/Glide targets whereas miR-279 negatively controls Gcm/Glide expression (ii) identify a novel cell population, peritracheal cells, expressing and requiring Gcm/Glide. Peritracheal cells are non-neuronal neurosecretory cells that are essential in ecdysis. In addition to emphasizing the importance of following the distribution and the activity of endogenous proteins in vivo, this study provides new insights and a novel frame to understand the Gcm/Glide biology.

De novo DNA methyltransferase activity in colorectal cancer is directed towards H3K36me3 marked CpG islands.

The aberrant gain of DNA methylation at CpG islands is frequently observed in colorectal tumours and may silence the expression of tumour suppressors such as MLH1. Current models propose that these CpG islands are targeted by de novo DNA methyltransferases in a sequence-specific manner, but this has not been tested. Using ectopically integrated CpG islands, here we find that aberrantly methylated CpG islands are subject to low levels of de novo DNA methylation activity in colorectal cancer cells. By delineating DNA methyltransferase targets, we find that instead de novo DNA methylation activity is targeted primarily to CpG islands marked by the histone modification H3K36me3, a mark associated with transcriptional elongation. These H3K36me3 marked CpG islands are heavily methylated in colorectal tumours and the normal colon suggesting that de novo DNA methyltransferase activity at CpG islands in colorectal cancer is focused on similar targets to normal tissues and not greatly remodelled by tumourigenesis.

Gain-of-function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions.

DNA methylation and Polycomb are key factors in the establishment of vertebrate cellular identity and fate. Here we report de novo missense mutations in DNMT3A, which encodes the DNA methyltransferase DNMT3A. These mutations cause microcephalic dwarfism, a hypocellular disorder of extreme global growth failure. Substitutions in the PWWP domain abrogate binding to the histone modifications H3K36me2 and H3K36me3, and alter DNA methylation in patient cells. Polycomb-associated DNA methylation valleys, hypomethylated domains encompassing developmental genes, become methylated with concomitant depletion of H3K27me3 and H3K4me3 bivalent marks. Such de novo DNA methylation occurs during differentiation of Dnmt3aW326R pluripotent cells in vitro, and is also evident in Dnmt3aW326R/+ dwarf mice. We therefore propose that the interaction of the DNMT3A PWWP domain with H3K36me2 and H3K36me3 normally limits DNA methylation of Polycomb-marked regions. Our findings implicate the interplay between DNA methylation and Polycomb at key developmental regulators as a determinant of organism size in mammals.

Global regulation of heterochromatin spreading by Leo1.

Heterochromatin plays important roles in eukaryotic genome regulation. However, the repressive nature of heterochromatin combined with its propensity to self-propagate necessitates robust mechanisms to contain heterochromatin within defined boundaries and thus prevent silencing of expressed genes. Here we show that loss of the PAF complex (PAFc) component Leo1 compromises chromatin boundaries, resulting in invasion of heterochromatin into flanking euchromatin domains. Similar effects are seen upon deletion of other PAFc components, but not other factors with related functions in transcription-associated chromatin modification, indicating a specific role for PAFc in heterochromatin regulation. Loss of Leo1 results in reduced levels of H4K16 acetylation at boundary regions, while tethering of the H4K16 acetyltransferase Mst1 to boundary chromatin suppresses heterochromatin spreading in leo1Δ cells, suggesting that Leo1 antagonises heterochromatin spreading by promoting H4K16 acetylation. Our findings reveal a previously undescribed role for PAFc in regulating global heterochromatin distribution.