Application of Celluspots peptide arrays for the analysis of the binding specificity of epigenetic reading domains to modified histone tails

<p>Abstract</p> <p>Background</p> <p>Epigenetic reading domains are involved in the regulation of gene expression and chromatin state by interacting with histones in a post-translational modification specific manner. A detailed knowledge of the target modifications of reading domains, including enhancing and inhibiting secondary modifications, will lead to a better understanding of the biological signaling processes mediated by reading domains.</p> <p>Results</p> <p>We describe the application of Celluspots peptide arrays which contain 384 histone peptides carrying 59 post translational modifications in different combinations as an inexpensive, reliable and fast method for initial screening for specific interactions of reading domains with modified histone peptides. To validate the method, we tested the binding specificities of seven known epigenetic reading domains on Celluspots peptide arrays, viz. the HP1ß and MPP8 Chromo domains, JMJD2A and 53BP1 Tudor domains, Dnmt3a PWWP domain, Rag2 PHD domain and BRD2 Bromo domain. In general, the binding results agreed with literature data with respect to the primary specificity of the reading domains, but in almost all cases we obtained additional new information concerning the influence of secondary modifications surrounding the target modification.</p> <p>Conclusions</p> <p>We conclude that Celluspots peptide arrays are powerful screening tools for studying the specificity of putative reading domains binding to modified histone peptides.</p

Targeted epigenome editing of an endogenouslocus with chromatin modifiers is not stably maintained

Background: DNA methylation and histone 3 lysine 9 (H3K9) methylation are considered as epigenetic marks that can be inherited through cell divisions. To explore the functional consequences and stability of these modifications, we employed targeted installment of DNA methylation and H3K9 methylation in the vascular endothelial growth factor A (VEGF-A) promoter using catalytic domains of DNA or H3K9 methyltransferases that are fused to a zinc finger protein which binds a site in the VEGF-A promoter. Results: Expression of the targeted DNA and H3K9 methyltransferases caused dense deposition of DNA methylation or H3K9 di- and trimethylation in the promoter of VEGF-A and downregulation of VEGF-A gene expression. We did not observe positive feedback between DNA methylation and H3K9 methylation. Upon loss of the targeted methyltransferases from the cells, the epigenetic marks, chromatin environment, and gene expression Levels returned to their original state, indicating that both methylation marks were not stably propagated after their installment. Conclusions: The clear anti-correlation between DNA or H3K9 methylation and gene expression suggests a direct role of these marks in transcriptional control. The lack of maintenance of the transiently induced silenced chromatin state suggests that the stability of epigenetic signaling is based on an epigenetic network consisting of several molecular marks. Therefore, for stable reprogramming, either multivalent deposition of functionally related epigenetic marks or longer-lasting trigger stimuli might be necessary

Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.

DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20-30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling

H3K14ac is linked to methylation of H3K9 by the triple Tudor domain of SETDB1

SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the subnuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions

Quality of histone modification antibodies undermines chromatin biology research [version 2; referees: 1 approved, 2 approved with reservations]

Histone post-translational modification (PTM) antibodies are essential research reagents in chromatin biology. However, they suffer from variable properties and insufficient documentation of quality. Antibody manufacturers and vendors should provide detailed lot-specific documentation of quality, rendering further quality checks by end-customers unnecessary. A shift from polyclonal antibodies towards sustainable reagents like monoclonal or recombinant antibodies or histone binding domains would help to improve the reproducibility of experimental work in this field

Trio Clinical Exome Sequencing in a Patient With Multicentric Carpotarsal Osteolysis Syndrome: First Case Report in the Balkans

Exome sequencing can interrogate thousands of genes simultaneously and it is becoming a first line diagnostic tool in genomic medicine. Herein, we applied trio clinical exome sequencing (CES) in a patient presenting with undiagnosed skeletal disorder, minor facial abnormalities, and kidney hypoplasia; her parents were asymptomatic. Testing the proband and her parents led to the identification of a de novo mutation c.188C&gt;T (p.Pro63Leu) in the MAFB gene, which is known to cause multicentric carpotarsal osteolysis syndrome (MCTO). The c.188C&gt;T mutation lies in a hotspot amino acid stretch within the transactivation domain of MAFB, which is a negative regulator of RANKL-induced osteoclastogenesis. MCTO is an extremely rare autosomal dominant (AD) disorder that typically arises spontaneously and causes carpotarsal osteolysis, often followed by nephropathy. To the best of our knowledge, this is the first study reporting genetically diagnosed MCTO in the Balkans

MOESM1 of Application of recombinant TAF3 PHD domain instead of anti-H3K4me3 antibody

Additional file 1. Supplementary figures and table

MOESM1 of Application of dual reading domains as novel reagents in chromatin biology reveals a new H3K9me3 and H3K36me2/3 bivalent chromatin state

Additional file 1: Figure S1. Analysis of the coexistence of H3K9me3 and H3K36me2/3 in one H3 molecule by mass spectrometry. Figure S2. Control experiments related to Fig. 2. Figure S3. Quality control of double peptide spot arrays. Figure S4. Definition of chromatin states in HepG2 cells based on H3K9me3 and H3K36me2/3 CIDOP-seq signals using 1 kb bins. Figure S5. Definition of chromatin states based on Ernst et al. 2011. Figure S6. Annotated sequence of the D3PWWP-M8Chromo double domain. Table S1. GO analysis of low expression gene clusters shown in Fig. 6c. Table S2. GO analysis of the clusters of genes regulated by weak enhancers-2 shown in Fig. 7b. Table S3. GO analysis of the clusters of genes regulated by strong enhancers-2 shown in Fig. 7c. Table S4. Sequences of primers used in Fig. 5 for CIDOP-qPCR. Table S5. Sequences of primers used in Fig. 8b for CIDOP-qPCR