Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

Extensive studies of the structure–function relationship of antibodies have established that conventional immunoglobulins contain two copies of the antigen-binding fragment (Fab), each of which serves as an autonomous and complete unit for recognizing an antigen. In this paper, we report a previously unidentified mode of antibody–antigen recognition, dubbed “antigen clasping,” where two antigen-binding sites cooperatively clasp one antigen, and the design of a long-neck antibody format that facilitates antigen clasping. Antigen clasping led to recombinant antibodies for histone posttranslational modifications with extraordinarily high specificity, valuable tools for epigenetic research. This study substantially broadens the long-standing paradigm for antibody–antigen recognition

An Interactive Database for the Assessment of Histone Antibody Specificity

Access to high quality antibodies is a necessity for the study of histones and their posttranslational modifications (PTMs). Here we debut The Histone Antibody Specificity Database (http://www.histoneantibodies.com), an online and expanding resource cataloguing the behavior of widely used commercially available histone antibodies by peptide microarray. This interactive web portal provides a critical resource to the biological research community who routinely use these antibodies as detection reagents for a wide range of applications

Epigenetic homogeneity in histone methylation underlies sperm programming for embryonic transcription

Abstract: Sperm contributes genetic and epigenetic information to the embryo to efficiently support development. However, the mechanism underlying such developmental competence remains elusive. Here, we investigated whether all sperm cells have a common epigenetic configuration that primes transcriptional program for embryonic development. Using calibrated ChIP-seq, we show that remodelling of histones during spermiogenesis results in the retention of methylated histone H3 at the same genomic location in most sperm cell. This homogeneously methylated fraction of histone H3 in the sperm genome is maintained during early embryonic replication. Such methylated histone fraction resisting post-fertilisation reprogramming marks developmental genes whose expression is perturbed upon experimental reduction of histone methylation. A similar homogeneously methylated histone H3 fraction is detected in human sperm. Altogether, we uncover a conserved mechanism of paternal epigenetic information transmission to the embryo through the homogeneous retention of methylated histone in a sperm cells population

Sequence deeper without sequencing more: Bayesian resolution of ambiguously mapped reads

Next-generation sequencing (NGS) has transformed molecular biology and contributed to many seminal insights into genomic regulation and function. Apart from whole-genome sequencing, an NGS workflow involves alignment of the sequencing reads to the genome of study, after which the resulting alignments can be used for downstream analyses. However, alignment is complicated by the repetitive sequences; many reads align to more than one genomic locus, with 15–30% of the genome not being uniquely mappable by short-read NGS. This problem is typically addressed by discarding reads that do not uniquely map to the genome, but this practice can lead to systematic distortion of the data. Previous studies that developed methods for handling ambiguously mapped reads were often of limited applicability or were computationally intensive, hindering their broader usage. In this work, we present SmartMap: an algorithm that augments industry-standard aligners to enable usage of ambiguously mapped reads by assigning weights to each alignment with Bayesian analysis of the read distribution and alignment quality. SmartMap is computationally efficient, utilizing far fewer weighting iterations than previously thought necessary to process alignments and, as such, analyzing more than a billion alignments of NGS reads in approximately one hour on a desktop PC. By applying SmartMap to peak-type NGS data, including MNase-seq, ChIP-seq, and ATAC-seq in three organisms, we can increase read depth by up to 53% and increase the mapped proportion of the genome by up to 18% compared to analyses utilizing only uniquely mapped reads. We further show that SmartMap enables the analysis of more than 140,000 repetitive elements that could not be analyzed by traditional ChIP-seq workflows, and we utilize this method to gain insight into the epigenetic regulation of different classes of repetitive elements. These data emphasize both the dangers of discarding ambiguously mapped reads and their power for driving biological discovery.</p

Non-canonical H3k79me2-dependent pathways promote the survival of MLL-rearranged leukemia

MLL-rearranged leukemia depends on H3K79 methylation. Depletion of this transcriptionally activating mark by DOT1L deletion or high concentrations of the inhibitor pinometostat downregulates HOXA9 and MEIS1, and consequently reduces leukemia survival. Yet, some MLL-rearranged leukemias are inexplicably susceptible to low-dose pinometostat, far below concentrations that downregulate this canonical proliferation pathway. In this context, we define alternative proliferation pathways that more directly derive from H3K79me2 loss. By ICeChIP-seq, H3K79me2 is markedly depleted at pinometostat-downregulated and MLL-fusion targets, with paradoxical increases of H3K4me3 and loss of H3K27me3. Although downregulation of polycomb components accounts for some of the proliferation defect, transcriptional downregulation of FLT3 is the major pathway. Loss-of-FLT3-function recapitulates the cytotoxicity and gene expression consequences of low-dose pinometostat, whereas overexpression of constitutively active STAT5A, a target of FLT3-ITD-signaling, largely rescues these defects. This pathway also depends on MLL1, indicating combinations of DOT1L, MLL1 and FLT3 inhibitors should be explored for treating FLT3-mutant leukemia

Merging short and stranded long reads improves transcript assembly

Long-read RNA sequencing has arisen as a counterpart to short-read sequencing, with the potential to capture full-length isoforms, albeit at the cost of lower depth. Yet this potential is not fully realized due to inherent limitations of current long-read assembly methods and underdeveloped approaches to integrate short-read data. Here, we critically compare the existing methods and develop a new integrative approach to characterize a particularly challenging pool of low-abundance long noncoding RNA (lncRNA) transcripts from short- and long-read sequencing in two distinct cell lines. Our analysis reveals severe limitations in each of the sequencing platforms. For short-read assemblies, coverage declines at transcript termini resulting in ambiguous ends, and uneven low coverage results in segmentation of a single transcript into multiple transcripts. Conversely, long-read sequencing libraries lack depth and strand-of-origin information in cDNA-based methods, culminating in erroneous assembly and quantitation of transcripts. We also discover a cDNA synthesis artifact in long-read datasets that markedly impacts the identity and quantitation of assembled transcripts. Towards remediating these problems, we develop a computational pipeline to “strand” long-read cDNA libraries that rectifies inaccurate mapping and assembly of long-read transcripts. Leveraging the strengths of each platform and our computational stranding, we also present and benchmark a hybrid assembly approach that drastically increases the sensitivity and accuracy of full-length transcript assembly on the correct strand and improves detection of biological features of the transcriptome. When applied to a challenging set of under-annotated and cell-type variable lncRNA, our method resolves the segmentation problem of short-read sequencing and the depth problem of long-read sequencing, resulting in the assembly of coherent transcripts with precise 5’ and 3’ ends. Our workflow can be applied to existing datasets for superior demarcation of transcript ends and refined isoform structure, which can enable better differential gene expression analyses and molecular manipulations of transcripts

Quantitative and Structural Assessment of Histone Methyllysine Analogue Engagement by Cognate Binding Proteins Reveals Affinity Decrements Relative to Those of Native Counterparts

Methyllysine analogues (MLAs), furnished by aminoethylation of engineered cysteine residues, are widely used surrogates of histone methyllysine and are considered to be effective proxies for studying these epigenetic marks in vitro. Here we report the first structure of a trimethyllysine MLA histone in complex with a protein binding partner, quantify the thermodynamic distinctions between MLAs and their native methyllysine counterparts, and demonstrate that these differences can compromise qualitative interpretations of binding at the nucleosome level. Quantitative measurements with two methyllysine binding protein modules reveal substantial affinity losses for the MLA peptides versus the corresponding native methyllysine species in both cases, although the thermodynamic underpinnings are distinct. MLA and methyllysine adopt distinct conformational geometries when in complex with the BPTF PHD finger, a well-established H3K4me3 binding partner. In this case, an ∼13-fold <i>K</i>_d difference at the peptide level translates to nucleosomal affinities for MLA analogues that fall outside of the detectable range in a pull-down format, whereas the methyllysine species installed by native chemical ligation demonstrates robust binding. Thus, despite their facile production and commercial availability, there is a significant caveat of potentially altered binding affinity when MLAs are used in place of native methyllysine residues

Chromatin-enriched RNAs mark active and repressive cis-regulation: An analysis of nuclear RNA-seq.

Long noncoding RNAs (lncRNAs) localize in the cell nucleus and influence gene expression through a variety of molecular mechanisms. Chromatin-enriched RNAs (cheRNAs) are a unique class of lncRNAs that are tightly bound to chromatin and putatively function to locally cis-activate gene transcription. CheRNAs can be identified by biochemical fractionation of nuclear RNA followed by RNA sequencing, but until now, a rigorous analytic pipeline for nuclear RNA-seq has been lacking. In this study, we survey four computational strategies for nuclear RNA-seq data analysis and develop a new pipeline, Tuxedo-ch, which outperforms other approaches. Tuxedo-ch assembles a more complete transcriptome and identifies cheRNA with higher accuracy than other approaches. We used Tuxedo-ch to analyze benchmark datasets of K562 cells and further characterize the genomic features of intergenic cheRNA (icheRNA) and their similarity to enhancer RNAs (eRNAs). We quantify the transcriptional correlation of icheRNA and adjacent genes and show that icheRNA is more positively associated with neighboring gene expression than eRNA or cap analysis of gene expression (CAGE) signals. We also explore two novel genomic associations of cheRNA, which indicate that cheRNAs may function to promote or repress gene expression in a context-dependent manner. IcheRNA loci with significant levels of H3K9me3 modifications are associated with active enhancers, consistent with the hypothesis that enhancers are derived from ancient mobile elements. In contrast, antisense cheRNA (as-cheRNA) may play a role in local gene repression, possibly through local RNA:DNA:DNA triple-helix formation

Chromatin-enriched RNAs mark active and repressive cis-regulation: An analysis of nuclear RNA-seq

Long noncoding RNAs (lncRNAs) localize in the cell nucleus and influence gene expression through a variety of molecular mechanisms. Chromatin-enriched RNAs (cheRNAs) are a unique class of lncRNAs that are tightly bound to chromatin and putatively function to locally cis-activate gene transcription. CheRNAs can be identified by biochemical fractionation of nuclear RNA followed by RNA sequencing, but until now, a rigorous analytic pipeline for nuclear RNA-seq has been lacking. In this study, we survey four computational strategies for nuclear RNA-seq data analysis and develop a new pipeline, Tuxedo-ch, which outperforms other approaches. Tuxedo-ch assembles a more complete transcriptome and identifies cheRNA with higher accuracy than other approaches. We used Tuxedo-ch to analyze benchmark datasets of K562 cells and further characterize the genomic features of intergenic cheRNA (icheRNA) and their similarity to enhancer RNAs (eRNAs). We quantify the transcriptional correlation of icheRNA and adjacent genes and show that icheRNA is more positively associated with neighboring gene expression than eRNA or cap analysis of gene expression (CAGE) signals. We also explore two novel genomic associations of cheRNA, which indicate that cheRNAs may function to promote or repress gene expression in a context-dependent manner. IcheRNA loci with significant levels of H3K9me3 modifications are associated with active enhancers, consistent with the hypothesis that enhancers are derived from ancient mobile elements. In contrast, antisense cheRNA (as-cheRNA) may play a role in local gene repression, possibly through local RNA:DNA:DNA triple-helix formation