Biology subjects

Kaufman, P. D.

Publications and source records attributed to Kaufman, P. D..

9 recordsLinked to original sources

bioRxiv2026

OTTR-CLASH: improved biochemical and bioinformatic identification of Argonaute 2-mediated microRNA-target RNA interactions

Various methods have detected miRNA-target interactions via immunoprecipitation of UV-crosslinked Argonaute ribonucleoprotein complexes, followed by intermolecular ligation of bound miRNAs to target strands, forming chimeric RNAs. To date, these methods have relied on conventional viral reverse transcriptases (RTs) to generate cDNAs for sequencing. However, crosslinked RNAs often retain adducts after purification, which can make them poor templates for viral RTs. Here, we adapted OTTR (Ordered Two-Template Relay) techniques to generate cDNAs from Ago2-bound RNAs. OTTR makes use of a modified retroelement-encoded RT, which is strongly processive even on templates with modifications or adducts. We show that this "OTTR-CLASH" method increases the frequency of generating chimeric RNAs compared to previous methods. We also developed an improved bioinformatic pipeline for analysis of these data, and we use this to catalog miRNA-target interactions not previously described in the literature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/738487v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@16d62f8org.highwire.dtl.DTLVardef@7c8da4org.highwire.dtl.DTLVardef@13727fborg.highwire.dtl.DTLVardef@21f443_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

A novel family of lncRNAs relate facioscapulohumeral muscular dystrophy to nucleolar architecture and protein synthesis rate

Facioscapulohumeral muscular dystrophy (FSHD) is a hereditary myopathy linked to deletions of the tandemly arrayed D4Z4 macrosatellite repeats at human chromosome 4q35. These deletions accompany local chromatin changes and the anomalous expression of nearby transcripts FRG2A, DBET, and D4Z4. We discovered that FRG2A is one member of a family of long non-coding RNAs (lncRNAs) expressed at elevated levels in skeletal muscle cells with distinct amounts detected in individual patients. We found that FRG2A lncRNA preferentially associates with rDNA sequences and centromeres and promotes the three-dimensional association of centromeres with the nucleolar periphery in FSHD cells. Furthermore, we demonstrate that the elevated FRG2A expression in cells from FSHD patients reduces rDNA transcription and global protein synthesis. Our results frame an entirely unanticipated new disease model in which elevated lncRNAs levels mediated by deletions of D4Z4 macrosatellite repeats leads to a diminished protein synthesis capacity, thereby contributing to muscle wasting.

molecular biology↗

bioRxiv2024

Post-transcriptional RNA stabilization of telomere-proximal RNAs FRG2, DBET, D4Z4 at human 4q35 in response to genotoxic stress and D4Z4 macrosatellite repeat length.

BackgroundReduced copy number of the D4Z4 macrosatellite at human chromosome 4q35 is associated with facioscapulohumeral muscular dystrophy (FSHD). A pervasive idea is that chromatin alterations at the 4q35 locus following D4Z4 repeat unit deletion lead to disease via inappropriate expression of nearby genes. Here, we sought to analyze transcription and chromatin characteristics across 4q35 and how these are affected by D4Z4 deletions and exogenous stresses. ResultsWe found that the 4q subtelomere is subdivided into discrete domains, each with characteristic chromatin features associated with distinct gene expression profiles. Centromere-proximal genes within 4q35 (ANT1, FAT1 and FRG1) display active histone marks at their promoters. In contrast, poised or repressed markings are present at telomere-proximal loci including FRG2, DBE-T and D4Z4. We discovered that these discrete domains undergo region-specific chromatin changes upon treatment with chromatin enzyme inhibitors or genotoxic drugs. We demonstrated that the 4q35 telomere-proximal FRG2, DBE-T and D4Z4-derived transcripts are induced upon DNA damage to levels inversely correlated with the D4Z4 repeat number, are stabilized through post-transcriptional mechanisms upon DNA damage, and are bound to chromatin. ConclusionOur study reveals unforeseen biochemical features of RNAs from clustered transcription units within the 4q35 subtelomere. Specifically, the FRG2, DBE-T and D4Z4-derived transcripts are chromatin-associated and are stabilized post-transcriptionally after induction by genotoxic stress. Remarkably, the extent of this response is modulated by the copy number of the D4Z4 repeats, raising new hypotheses about their regulation and function in human biology and disease.

genetics↗

bioRxiv2023

Nucleolus and centromere TSA-Seq reveals variable localization of heterochromatin in different cell types

Genome differential positioning within interphase nuclei remains poorly explored. We extended and validated TSA-seq to map genomic regions near nucleoli and pericentric heterochromatin in four human cell lines. Our study confirmed that smaller chromosomes localize closer to nucleoli but further deconvolved this by revealing a preference for chromosome arms below 36-46 Mbp in length. We identified two lamina associated domain subsets through their differential nuclear lamina versus nucleolar positioning in different cell lines which showed distinctive patterns of DNA replication timing and gene expression across all cell lines. Unexpectedly, active, nuclear speckle-associated genomic regions were found near typically repressive nuclear compartments, which is attributable to the close proximity of nuclear speckles and nucleoli in some cell types, and association of centromeres with nuclear speckles in hESCs. Our study points to a more complex and variable nuclear genome organization than suggested by current models, as revealed by our TSA-seq methodology.

cell biology↗

bioRxiv2020

The chromatin-binding domain of Ki-67 together with p53 protects human chromosomes from mitotic damage

Vertebrate mammals express a protein called Ki-67 which is most widely known as a clinically useful marker of highly proliferative cells. Previous studies of human cells indicated that acute depletion of Ki-67 can elicit a delay at the G1/S boundary of the cell cycle, dependent on induction of the checkpoint protein p21. Consistent with those observations, we show here that acute Ki-67 depletion causes hallmarks of DNA damage, and the damage occurs even in the absence of checkpoint signaling. This damage is not observed in cells traversing S phase but is instead robustly detected in mitotic cells. The C-terminal chromatin binding domain of Ki-67 is necessary and sufficient to protect cells from this damage. We also observe synergistic effects when Ki-67 and p53 are simultaneously depleted, resulting in increased levels of chromosome bridges at anaphase, followed by the appearance of micronuclei. Therefore, these studies identify the C-terminus of Ki-67 as an important module for genome stability.

cell biology↗

bioRxiv2019

Distinct features of nucleolus-associated domains in mouse embryonic stem cells

BackgroundHeterochromatin in eukaryotic interphase cells frequently localizes to the nucleolar periphery (nucleolus-associated domains, NADs) and the nuclear lamina (lamina-associated domains, LADs). Gene expression in somatic cell NADs is generally low, but NADs have not been characterized in mammalian stem cells.\n\nResultsHere, we generated the first genome-wide map of NADs in mouse embryonic stem cells (mESCs) via deep sequencing of chromatin associated with biochemically-purified nucleoli. As we had observed in mouse embryonic fibroblasts (MEFs), the large Type I subset of NADs overlaps with constitutive LADs and is enriched for features of constitutive heterochromatin, including late replication timing and low gene density and expression levels. Conversely, the Type II NAD subset overlaps with loci that are not lamina-associated, but in mESCs, Type II NADs are much less abundant than in MEFs. mESC NADs are also much less enriched in H3K27me3 modified regions than are NADs in MEFs. Additionally, comparision of MEF and mESC NADs revealed enrichment of developmentally regulated genes in cell type-specific NADs. Together, these data indicate that NADs are a developmentally dynamic component of heterochromatin.\n\nConclusionsThese studies implicate association with the nucleolar periphery as a mechanism for developmentally-regulated gene silencing, and will facilitate future studies of NADs during mESC differentiation.

genomics↗

bioRxiv2018

Two Contrasting Classes of Nucleolus-Associated Domains in Mouse Fibroblast Heterochromatin

In interphase eukaryotic cells, almost all heterochromatin is located adjacent to the nucleolus or to the nuclear lamina, thus defining Nucleolus-Associated Domains (NADs) and Lamina-Associated Domains (LADs), respectively. Here, we determined the first genome-scale map of murine NADs in mouse embryonic fibroblasts (MEFs) via deep sequencing of chromatin associated with purified nucleoli. We developed a Bioconductor package called NADfinder and demonstrated that it identifies NADs more accurately than other peak-calling tools, due to its critical feature of chromosome-level local baseline correction. We detected two distinct classes of NADs. Type I NADs associate frequently with both the nucleolar periphery and with the nuclear lamina, and generally display characteristics of constitutive heterochromatin, including late DNA replication, enrichment of H3K9me3 and little gene expression. In contrast, Type II NADs associate with nucleoli but do not overlap with LADs. Type II NADs tend to replicate earlier, display greater gene expression, and are more often enriched in H3K27me3 than Type I NADs. The nucleolar associations of both classes of NADs were confirmed via DNA-FISH, which also detected Type I but not Type II probes enriched at the nuclear lamina. Interestingly, Type II NADs are enriched in distinct gene classes, notably factors important for differentiation and development. In keeping with this, we observed that a Type II NAD is developmentally regulated, present in MEFs but not in undifferentiated embryonic stem (ES) cells.

genomics↗

bioRxiv2017

A synthetic biology approach to probing nucleosome symmetry

The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell.

molecular biology↗

bioRxiv2017

Ki-67 Contributes To Normal Cell Cycle Progression And Inactive X Heterochromatin In p21 Checkpoint-Proficient Human Cells

Ki-67 protein is widely used as a tumor proliferation marker. However, whether Ki-67 affects cell cycle progression has been controversial. Here, we demonstrate that depletion of Ki-67 in human hTERT-RPE1, WI-38, IMR90, hTERT-BJ cell lines and primary fibroblast cells slowed entry into S phase and coordinately downregulated genes related to DNA replication. Some gene expression changes were partially relieved in Ki-67-depleted hTERT-RPE1 cells by co-depletion of the Rb checkpoint protein, but more thorough suppression of the transcriptional and cell cycle defects was observed upon depletion of cell cycle inhibitor p21. Notably, induction of p21 upon depletion of Ki-67 was a consistent hallmark of cell types in which transcription and cell cycle distribution were sensitive to Ki-67; these responses were absent in cells that did not induce p21. Furthermore, upon Ki-67 depletion, a subset of inactive x (Xi) chromosomes in female hTERT-RPE1 cells displayed several features of compromised heterochromatin maintenance, including decreased H3K27me3 and H4K20me1 labeling. These chromatin alterations were limited to Xi chromosomes localized away from the nuclear lamina and were not observed in checkpoint-deficient 293T cells. Altogether, our results indicate that Ki-67 integrates normal S phase progression and Xi heterochromatin maintenance in p21 checkpoint-proficient human cells.

molecular biology↗