Structure and regulation of ZCCHC4 in m6A-methylation of 28S rRNA.

N6-methyladenosine (m6A) modification provides an important epitranscriptomic mechanism that critically regulates RNA metabolism and function. However, how m6A writers attain substrate specificities remains unclear. We report the 3.1 Å-resolution crystal structure of human CCHC zinc finger-containing protein ZCCHC4, a 28S rRNA-specific m6A methyltransferase, bound to S-adenosyl-L-homocysteine. The methyltransferase (MTase) domain of ZCCHC4 is packed against N-terminal GRF-type and C2H2 zinc finger domains and a C-terminal CCHC domain, creating an integrated RNA-binding surface. Strikingly, the MTase domain adopts an autoinhibitory conformation, with a self-occluded catalytic site and a fully-closed cofactor pocket. Mutational and enzymatic analyses further substantiate the molecular basis for ZCCHC4-RNA recognition and a role of the stem-loop structure within substrate in governing the substrate specificity. Overall, this study unveils unique structural and enzymatic characteristics of ZCCHC4, distinctive from what was seen with the METTL family of m6A writers, providing the mechanistic basis for ZCCHC4 modulation of m6A RNA methylation

Tudor: a versatile family of histone methylation ‘readers’

The Tudor domain comprises a family of motifs that mediate protein-protein interactions required for various DNA-templated biological processes. Emerging evidence demonstrates a versatility of the Tudor family domains by identifying their specific interactions to a wide variety of histone methylation marks. Here, we discuss novel functions of a number of Tudor-containing proteins (including JMJD2A, 53BP1, SGF29, Spindlin1, UHRF1, PHF1, PHF19 and SHH1) in ‘reading’ unique methylation events on histones in order to facilitate DNA damage repair or regulate transcription. This review covers our recent understanding of the molecular bases for histone-Tudor interactions and their biological outcomes. As deregulation of Tudor-containing proteins is associated with certain human disorders, pharmacological targeting of Tudor interactions could provide new avenues for therapeutic intervention

BPTF Is Essential for T Cell Homeostasis and Function

Bromodomain PHD Finger Transcription Factor (BPTF), a ubiquitously expressed ATP-dependent chromatin-remodeling factor, is critical for epigenetically regulating DNA accessibility and gene expression. While BPTF is important for the development of thymocytes, its function in mature T cells remains largely unknown. By specifically deleting BPTF from late DN3/DN4 stage of developing T cells, we found that BPTF was critical for the homeostasis of T cells via a cell intrinsic manner. In addition, BPTF was essential for the maintenance and function of Treg cells. Treg cell-specific BPTF deletion led to reduced Foxp3 expression, increased lymphocyte infiltration in the non-lymphoid organs and a systemic autoimmune syndrome. These findings therefore reveal a vital role for BPTF in T and Treg cell function and immune homeostasis

R-loops: formation, function, and relevance to cell stress

Exposure of genomic, single-stranded DNA (ssDNA) during transcription and replication creates opportunities for the formation of inappropriate secondary structures. Cells manage this exposure by using topoisomerases and helicases to reduce the inherent topological stress that arises from unwinding the double helix and by coating ssDNA with protective protein complexes. Interestingly, specific DNA-RNA hybrids, known as R-loops, form during transcription and exist in homeostasis throughout the genomes of prokaryotes and eukaryotes. These hybrids nucleate from guanine rich clusters in the template strand and extend across GC rich spans of transcribed genes. In vivo regulatory functions have evolved from R-loops, including regulation of gene expression and telomere lengthening. However, they also exist as a form of stress, particularly when replication forks collide with the transcription machinery. New methodologies and models are being developed to delineate the biology of R-loops, including those related to cell stress-based diseases like cancer. As accumulation of R-loops is associated with disease, targeting molecular pathways that regulate their formation or removal could provide new avenues for therapeutic intervention. This review covers recent understandings of the molecular basis for R-loop formation, removal, and biological outcomes in the context of cellular stress

Structural basis for DNMT3A-mediated de novo DNA methylation.

DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) at cytosines is essential for genome regulation and development. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-ångström crystal structure of the DNMT3A-DNMT3L-DNA complex in which two DNMT3A monomers simultaneously attack two cytosine-phosphate-guanine (CpG) dinucleotides, with the target sites separated by 14 base pairs within the same DNA duplex. The DNMT3A-DNA interaction involves a target recognition domain, a catalytic loop, and DNMT3A homodimeric interface. Arg836 of the target recognition domain makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Haematological cancer-associated somatic mutations of the substrate-binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of haematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its aetiological link to human disease

Targeting EZH2 and PRC2 dependence as novel anticancer therapy

Distinctive patterns of chromatin modification control gene expression and define cellular identity during development and cell differentiation. Polycomb repressive complex 2 (PRC2), the sole mammalian enzymatic complex capable of establishing gene-repressive high-degree methylation of histone H3 at lysine 27 (H3K27), plays crucial roles in regulation of normal and malignant hematopoiesis. Recently, increasing evidence has indicated that recurrent gain-of-function mutation and overexpression of EZH2, the catalytic subunit of PRC2, drive and promote malignant transformation such as B-cell lymphomagenesis, providing a rationale for PRC2 inhibition as a novel anticancer strategy. Here, we summarize the recently developed strategies for inhibition of PRC2, which include a series of highly specific, highly potent, small-molecule inhibitors of EZH2 and EZH1, an EZH2-related methyltransferase. PRC2 establishes functional crosstalk with numerous epigenetic machineries during dynamic regulation of gene transcription. Perturbation of such functional crosstalk caused by genetic events observed in various hematologic cancers, such as inactivation of SNF5 and somatic mutation of UTX, confers PRC2 dependence, thus rendering an increased sensitivity to PRC2 inhibition. We discuss our current understanding of EZH2 somatic mutations frequently found in B-cell lymphomas and recurrent mutations in various other epigenetic regulators as novel molecular predictors and determinants of PRC2 sensitivity. As recent advances have indicated a critical developmental or tumor-suppressive role for PRC2 and EZH2 in various tissue types, we discuss concerns over potentially toxic or even adverse effects associated with EZH2/1 inhibition in certain biological contexts or on cancer genetic background. Collectively, inhibition of PRC2 catalytic activity has emerged as a promising therapeutic intervention for the precise treatment of a range of genetically defined hematologic malignancies and can be potentially applied to a broader spectrum of human cancers that bear similar genetic and epigenetic characteristics. Published by Elsevier Inc. on behalf of ISEH - International Society for Experimental Hematology

WebTraceMiner: a web service for processing and mining EST sequence trace files

Expressed sequence tags (ESTs) remain a dominant approach for characterizing the protein-encoding portions of various genomes. Due to inherent deficiencies, they also present serious challenges for data quality control. Before GenBank submission, EST sequences are typically screened and trimmed of vector and adapter/linker sequences, as well as polyA/T tails. Removal of these sequences presents an obstacle for data validation of error-prone ESTs and impedes data mining of certain functional motifs, whose detection relies on accurate annotation of positional information for polyA tails added posttranscriptionally. As raw DNA sequence information is made increasingly available from public repositories, such as NCBI Trace Archive, new tools will be necessary to reanalyze and mine this data for new information. WebTraceMiner (www.conifergdb.org/software/wtm) was designed as a public sequence processing service for raw EST traces, with a focus on detection and mining of sequence features that help characterize 3′ and 5′ termini of cDNA inserts, including vector fragments, adapter/linker sequences, insert-flanking restriction endonuclease recognition sites and polyA or polyT tails. WebTraceMiner complements other public EST resources and should prove to be a unique tool to facilitate data validation and mining of error-prone ESTs (e.g. discovery of new functional motifs)

An Allosteric Interaction Links USP7 to Deubiquitination and Chromatin Targeting of UHRF1

The protein stability and chromatin functions of UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) are regulated in a cell-cycle-dependent manner. We report a structural characterization of the complex between UHRF1 and the deubiquitinase USP7. The first two UBL domains of USP7 bind to the polybasic region (PBR) of UHRF1, and this interaction is required for the USP7-mediated deubiquitination of UHRF1. Importantly, we find that the USP7-binding site of the UHRF1 PBR overlaps with the region engaging in an intramolecular interaction with the N-terminal tandem Tudor domain (TTD). We show that the USP7-UHRF1 interaction perturbs the TTD-PBR interaction of UHRF1, thereby shifting the conformation of UHRF1 from a TTD- occluded state to a state open for multivalent histone binding. Consistently, introduction of a USP7-interaction-defective mutation to UHRF1 significantly reduces its chromatin association. Together, these results link USP7 interaction to the dynamic deubiquitination and chromatin association of UHRF1

Epigenetic Perturbations by Arg882-Mutated DNMT3A Potentiate Aberrant Stem Cell Gene-Expression Program and Acute Leukemia Development

DNA methyltransferase 3A (DNMT3A) is frequently mutated in hematological cancers; however, the underlying oncogenic mechanism remains elusive. Here, we report that DNMT3A mutational hotspot at Arg882 (DNMT3AR882H) cooperates with NRAS mutation to transform hematopoietic stem/progenitor cells and induce acute leukemia development. Mechanistically, DNMT3AR882H directly binds to and potentiates transactivation of stemness genes critical for leukemogenicity including Meis1, Mn1 and Hoxa gene cluster. DNMT3AR882H induces focal epigenetic alterations, including CpG hypomethylation and concurrent gain of active histone modifications, at cis-regulatory elements such as enhancers to facilitate gene transcription. CRISPR/Cas9-mediated ablation of a putative Meis1 enhancer carrying DNMT3AR882H-induced DNA hypomethylation impairs Meis1 expression. Importantly, DNMT3AR882H-induced gene expression programs can be repressed through Dot1l inhibition, providing an attractive therapeutic strategy for DNMT3A-mutated leukemias

Land rebound after banning deep groundwater extraction in Changzhou, China

More than 30 years groundwater overdraft had resulted in hydraulic head declined from near the ground surface to 85 m deep in the second confined aquifer (CA2) in Changzhou, and lead to regional land subsidence from 1970's to early 2000's. After banning deep groundwater extraction was banned in 2000, the hydraulic head of CA2 had recovered to 37.6 m in Changzhou by the end of 2013. Based on several stages first and second order leveling results and long term monitoring data from the multi-strata borehole extensometer station (BES), it was revealed that the land subsidence is attributed to the compression of both aquifers and aquitards in the porous aquifer system. The spatial characteristics of subsidence are related not only to hydraulic head pattern in the area, but also to the thickness and compressibility of different soil strata, and distance from the aquifer. Since banning deep groundwater extraction, the ground uplifted 37.22 mm (5.4% of the pre subsidence) at BES, Changzhou due to the hydraulic head recovering. Strata compression and rebound was recorded as: the upper most stratum, and the underlying aquitard of CA2, and the upper CA3 are still in the compression process, the lower CA3 layer and the upper most segment of aquitard of CA2 rebounded about 90% of the pre compression recorded since 1984, and the CA2 and its adjacent overlying aquitard rebounded 3.8%–9.7% of the pre compression. © 2017 Elsevier B.V