The NSD (nuclear receptor-binding SET domain protein) family encodes methyltransferases that are important in multiple aspects of development and disease. Perturbations in NSD family members can lead to Sotos syndrome and Wolf-Hirschhorn syndrome as well as cancers such as acute myeloid leukemia. Previous studies have implicated NSD1 (KMT3B) in transcription and methylation of histone H3 at lysine 36 (H3-K36), but its molecular mechanism in these processes remains largely unknown. Here we describe an NSD1 regulatory network in human cells. We show that NSD1 binds near various promoter elements and regulates multiple genes that appear to have a concerted role in various processes, such as cell growth/cancer, keratin biology, and bone morphogenesis. In particular, we show that NSD1 binding is concentrated upstream of gene targets such as the bone morphogenetic protein 4 (BMP4) and zinc finger protein 36 C3H type-like 1 (ZFP36L1/TPP). NSD1 regulates the levels of the various forms of methylation at H3-K36 primarily, but not exclusively, within the promoter proximal region occupied by NSD1. At BMP4 we find that this reduces the levels of RNAP II recruited to the promoter, suggesting a role for NSD1-dependent methylation in initiation. Interestingly, we also observe that the RNAP II molecules that lie within BMP4 have inappropriate persistence of serine-5 phosphorylation and reduced levels of serine-2 phosphorylation within the C-terminal domain (CTD) of the large subunit of RNAP II. Our findings indicate that NSD1 regulates RNAP II recruitment to BMP4, and failure to do so leads to reduced gene expression and abrogated levels of H3K36Me and CTD phosphorylation.elongation | initiation | C-terminal domain | histone code | ChIP on chip
Increasing evidence indicates that the post-translational modifications of the histone proteins play critical roles in all eukaryotic DNA-templated processes. To gain further biological insights into two of these modifications, the mono- and trimethylation of histone H4 lysine 20 (H4K20me1 and H4K20me3), ChIP-chip experiments were performed to identify the precise genomic regions on human chromosomes 21 and 22 occupied by these two modifications. Detailed analysis revealed that H4K20me1 was preferentially enriched within specific genes; most significantly between the first approximately 5% and 20% of gene bodies. In contrast, H4K20me3 was preferentially targeted to repetitive elements. Among genes enriched in H4K20me3, the modification was typically targeted to a small region approximately 1 kb upstream of transcription start. Our collective findings strongly suggest that H4K20me1 and H4K20me3 are both physically and functionally distinct. We next sought to determine the role of H4K20me1 in transcription since this has been controversial. Following the reduction of PR-Set7/Set8/KMT5a and H4K20me1 in cells by RNAi, all H4K20me1-associated genes analyzed displayed an approximately 2-fold increase in gene expression; H4K20me3-associated genes displayed no changes. Similar results were obtained using a catalytically dead dominant negative PR-Set7 indicating that H4K20me1, itself, is essential for the selective transcriptional repression of H4K20me1-associated genes. Furthermore, we determined that the H4K20me1-associated DNA sequences were sufficient to nucleate H4K20me1 and induce repression in vivo. Our findings reveal the molecular mechanisms of a mammalian transcriptional repressive pathway whereby the DNA sequences within specific gene bodies are sufficient to nucleate the monomethylation of H4K20 which, in turn, reduces gene expression by half.
BackgroundThe evolution of multicellularity is accompanied by the occurrence of differentiated tissues, of organismal developmental programs, and of mechanisms keeping the balance between proliferation and differentiation. Initially, the SET-domain proteins were associated exclusively with regulation of developmental genes in metazoa. However, finding of SET-domain genes in the unicellular yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe suggested that SET-domain proteins regulate a much broader variety of biological programs. Intuitively, it is expected that the numbers, types, and biochemical specificity of SET-domain proteins of multicellular versus unicellular forms would reflect the differences in their biology. However, comparisons across the unicellular and multicellular domains of life are complicated by the lack of knowledge of the ancestral SET-domain genes. Even within the crown group, different biological systems might use the epigenetic 'code' differently, adapting it to organism-specific needs. Simplifying the model, we undertook a systematic phylogenetic analysis of one monophyletic fungal group (Ascomycetes) containing unicellular yeasts, Saccharomycotina (hemiascomycetes), and a filamentous fungal group, Pezizomycotina (euascomycetes).ResultsSystematic analysis of the SET-domain genes across an entire eukaryotic phylum has outlined clear distinctions in the SET-domain gene collections in the unicellular and in the multicellular (filamentous) relatives; diversification of SET-domain gene families has increased further with the expansion and elaboration of multicellularity in animal and plant systems. We found several ascomycota-specific SET-domain gene groups; each was unique to either Saccharomycotina or Pezizomycotina fungi. Our analysis revealed that the numbers and types of SET-domain genes in the Saccharomycotina did not reflect the habitats, pathogenicity, mechanisms of sexuality, or the ability to undergo morphogenic transformations. However, novel genes have appeared for functions associated with the transition to multicellularity. Descendents of most of the SET-domain gene families found in the filamentous fungi could be traced in the genomes of extant animals and plants, albeit as more complex structural forms.ConclusionSET-domain genes found in the filamentous species but absent from the unicellular sister group reflect two alternative evolutionary events: deletion from the yeast genomes or appearance of novel structures in filamentous fungal groups. There were no Ascomycota-specific SET-domain gene families (i.e., absent from animal and plant genomes); however, plants and animals share SET-domain gene subfamilies that do not exist in the fungi. Phylogenetic and gene-structure analyses defined several animal and plant SET-domain genes as sister groups while those of fungal origin were basal to them. Plants and animals also share SET-domain subfamilies that do not exist in fungi.
This article will review the recent advances in the understanding of the role of epigenetic modifications and the promise of future epigenetic therapy in neurodegenerative dementias, including Alzheimer's disease and frontotemporal dementia.
PR-Set7/Set8/KMT5a is a chromatin-modifying enzyme that specifically monomethylates lysine 20 of histone H4 (H4K20me1). In this study we attempted to identify PR-Set7-interacting proteins reasoning that these proteins would provide important insights into the role of PR-Set7 in transcriptional regulation. Using an unbiased yeast two-hybrid approach, we discovered that PR-Set7 interacts with the UBC9 E2 SUMO conjugating enzyme. This interaction was confirmed in human cells and we demonstrated that PR-Set7 was preferentially modified with SUMO1 in vivo. Further in vitro studies revealed that UBC9 directly binds PR-Set7 proximal to the catalytic SET domain. Two putative SUMO consensus sites were identified in this region and both were capable of being SUMOylated in vitro. The absence of either or both SUMO sites did not perturb nuclear localization of PR-Set7. By employing whole genome expression arrays, we identified a panel of genes whose expression was significantly altered in the absence of PR-Set7. The vast majority of these genes displayed increased expression strongly suggesting that PR-Set7 predominantly functions as a transcriptional repressor. Importantly, the reduction of UBC9 resulted in the consistent derepression of several of these newly identified genes regulated by PR-Set7. Our findings indicate that direct interaction with UBC9 facilitates the repressive effects of PR-Set7 at specific target genes, most likely by SUMOylating PR-Set7.
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