Clustered PHD domains in KMT2/MLL proteins are attracted by H3K4me3 and H3 acetylation-rich active promoters and enhancers
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ABSTRACT:
PROVIDER: PRJNA771366 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16343752_1.fastq.gz | Fastqsanger.gz | |||
| SRR16343752_2.fastq.gz | Fastqsanger.gz | |||
| SRR16343753_1.fastq.gz | Fastqsanger.gz | |||
| SRR16343753_2.fastq.gz | Fastqsanger.gz | |||
| SRR16343754_1.fastq.gz | Fastqsanger.gz |
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Project description:Histone lysine-specific methyltransferase 2 (KMT2A-D) proteins, alternatively called mixed lineage leukemia (MLL1-4) proteins, mediate positive transcriptional memory. Acting as the catalytic subunits of human COMPASS-like complexes, KMT2A-D methylate H3K4 at promoters and enhancers. KMT2A-D contain understudied highly conserved triplets and a quartet of plant homeodomains (PHDs). Here, we show that all clustered (multiple) PHDs localize to the well-defined loci of H3K4me3 and H3 acetylation-rich active promoters and enhancers. Surprisingly, we observe little difference in binding pattern between PHDs from promoter-specific KMT2A-B and enhancer-specific KMT2C-D. Fusion of the KMT2A CXXC domain to the PHDs drastically enhances their preference for promoters over enhancers. Hence, the presence of CXXC domains in KMT2A-B, but not KMT2C-D, may explain the promoter/enhancer preferences of the full-length proteins. Importantly, targets of PHDs overlap with KMT2A targets and are enriched in genes involved in the cancer pathways. We also observe that PHDs of KMT2A-D are mutated in cancer, especially within conserved folding motifs (Cys4HisCys2Cys/His). The mutations cause a domain loss-of-function. Taken together, our data suggest that PHDs of KMT2A-D guide the full-length proteins to active promoters and enhancers, and thus play a role in positive transcriptional memory.
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Project description:Histone lysine-specfic methyltransferase 2 (KMT2A-D) proteins, alternatively called mixed lineage leukaemia (MLL1-4) proteins, mediate positive transcriptional memory. As the catalytic subunits of human COMPASS-like complexes, they methylate H3K4 at promoters and enhancers. KMT2A-D contain understudied highly conserved triplets and a quartet of plant homeodomains (PHDs). Here, we show that all clustered PHDs localise to the well-defined loci of H3K4me3 and H3 acetylation-rich active promoters and enhancers. Surprisingly, we observe little difference in binding pattern between PHDs from promoter-specific KMT2A-B and enhancer-specific KMT2C-D. Fusion of the KMT2A CXXC domain to the PHDs drastically enhances their preference for promoters over enhancers. Hence, the presence of CXXC domains in KMT2A-B, but not KMT2C-D, may explain the promoter/enhancer preferences of the full-length proteins. Importantly, targets of PHDs overlap with KMT2A targets and are enriched in genes involved in the cancer pathways. We also observe that PHDs of KMT2A-D are mutated in cancer, especially within conserved folding motifs (Cys4HisCys2Cys/His), which cause a domain loss-of-function. Taken together, our data suggests that PHDs of KMT2A-D guide the full-length proteins to active promoters and enhancers, and thus play a role in positive transcriptional memory.
2022-12-08 | GSE185921 | GEO
Project description:Application of recombinant Taf3 PHD domain instead of anti-H3K4me3 antibody in ChIP-seq-like experiments (CIDOP-seq)
2016-05-31 | E-MTAB-4103 | biostudies-arrayexpress
Project description:Mixed-lineage leukemia (MLL) maintains the expression of cellular memory genes during development, while leukemic MLL fusion proteins aberrantly maintain expression of hematopoietic stem cell program genes such as HOXA9 to cause leukemia. However, the molecular mechanism of gene activation is unclear. Here we show that only two functional modules are necessary and sufficient for target recognition: those that bind to non-methylated CpGs and di-/tri-methylated histone H3 lysine 36 (H3K36me2/3). An artificial protein composed of the two targeting modules and an interaction domain for AF4-family coactivators can functionally substitute for MLL fusion proteins. Because H3K36me2/3 markers are indicative of active transcription, MLL fusion proteins target previously active CpG-rich genes and activate transcription by recruiting coactivators thereto. Our results indicate that such chromatin context-dependent gene activation is the fundamental mechanism by which MLL fusion proteins maintain the expression of the cellular memory/hematopoietic stem cell program genes.
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Project description:The lysine-specific demethylase 2A gene (KDM2A) is ubiquitously expressed and its transcripts consist of several alternatively spliced forms, including KDM2A and the shorter form N782 that lacks the 3’ end encoding F-box and LRR. KDM2A binds to numerous CpG-rich genomic loci and regulates various cellular activities; however, the mechanism of the pleiotropic function is unknown. Here, we identify the mechanism of KDM2A played by its CXXC-PHD domain. KDM2A is necessary for a rapid proliferation of post-natal keratinocytes while its 3’ end eclipses the stimulatory effect. EGFP-N782 binds to chromatin together with the XRCC5/6 complex, and the CXXC-PHD domain regulates the CpG-rich IGFBPL1 promoter. In vitro, CXXC-PHD enhances binding of nuclear extract ORC3 to the CpG-rich promoter, but not to the AT-rich DIP2B promoter to which ORC3 binds constitutively. Furthermore, CXXC-PHD recruits 94 nuclear factors involved in replication, ribosome synthesis, and mitosis, including POLR1A to the IGFBPL1 promoter. This recruitment is unprecedented; however, the result suggests that these nuclear factors bind to their cognate loci, as substantiated by the result that CXXC-PHD recruits POLR1A to the rDNA promoter. We propose that CXXC-PHD promotes permissiveness for nuclear factors to interact, but involvement of the XRCC5/6 complex in the recruitment is undetermined.
2019-06-28 | PXD012941 | Pride
Project description:Application of recombinant PHD domain instead of anti-H3K4me3 antibody
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Project description:Chromosomal translocations involving the mixed lineage leukemia (MLL) gene are associated with aggressive acute lymphoid and myeloid leukemias. These translocations are restricted to an 8.3-kb breakpoint region resulting in fusion of amino terminal MLL sequences in frame to 1 of more than 60 different translocation partners. The translocations consistently delete the plant homeodomain (PHD) fingers and more carboxyl terminal MLL sequences. The function of the PHD fingers is obscure and their specific role in transformation has not been explored. Here we show that inclusion of the PHD fingers in the MLL fusion protein MLL-AF9 blocked immortalization of hematopoietic progenitors. Inclusion of 2 or more PHD fingers reduced association with the Hoxa9 locus and suppressed Hoxa9 up-regulation in hematopoietic progenitors. These data provide an explanation for why MLL translocation breakpoints exclude the PHD fingers and suggest a possible role for these domains in regulating the function of wild-type MLL.
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Project description:The PHD fingers of the human MLL and Drosophila trx proteins have strong amino acid sequence conservation but their function is unknown. We have determined that these fingers mediate homodimerization and binding of MLL to Cyp33, a nuclear cyclophilin. These two proteins interact in vitro and in vivo in mammalian cells and colocalize at specific nuclear subdomains. Overexpression of the Cyp33 protein in leukemia cells results in altered expression of HOX genes that are targets for regulation by MLL. These alterations are suppressed by cyclosporine and are not observed in cell lines that express a mutant MLL protein without PHD fingers. These results suggest that binding of Cyp33 to MLL modulates its effects on the expression of target genes.
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Project description:BackgroundHistone posttranslational modifications (PTMs) represent a focal point of chromatin regulation. The genome-wide and locus-specific distribution and the presence of distinct histone PTMs is most commonly examined with the application of histone PTM-specific antibodies. In spite of their central role in chromatin research, polyclonal antibodies suffer from disadvantages like batch-to-batch variability and insufficient documentation of their quality and specificity.ResultsTo mitigate some of the pitfalls of using polyclonal antibodies against H3K4me3, we successfully validated the application of a recombinant TAF3 PHD domain as anti-H3K4me3 affinity reagent in peptide array, western blot and ChIP-like experiments coupled with qPCR and deep sequencing.ConclusionsThe successful addition of the TAF3 PHD domain to the growing catalog of recombinant affinity reagents for histone PTMs could help to improve the reproducibility, interpretation and cross-laboratory validation of chromatin data.
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Project description:Control of gene expression depends on a complex and delicate balance of various posttranslational modifications of histones. However, the relevance of specific combinations of histone modifications is not fully defined. Downstream effector proteins recognize particular histone modifications and transduce this information into gene expression patterns. Methylation of histone H3 at lysine 4 (H3K4me) is a landmark of gene expression control in eukaryotes. Its recognition depends on the presence in the effector protein of a motif termed plant homeodomain (PHD) that specifically binds to H3K4me3. Here, we establish that Arabidopsis ORC1, the large subunit of the origin recognition complex involved in defining origins of DNA replication, functions as a transcriptional activator of a subset of genes, the promoters of which are preferentially bound by ORC1. Arabidopsis ORC1 contains a PHD and binds to H3K4me3. In addition to H4 acetylation, ORC1 binding correlates with increased H4K20me3 in the proximal promoter region of ORC1 targets. This suggests that H4K20me3, unlike in animal cells, is associated with transcriptional activation in Arabidopsis. Thus, our data provide a molecular basis for the opposite role of ORC1 in transcriptional activation in plants and repression in animals. Since only ORC1 proteins of plant species contain a PHD, we propose that plant ORC1 constitutes a novel class of H3K4me3 effector proteins characteristic of the plant kingdom.
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