Biology subjects

Rando, O. J.

Publications and source records attributed to Rando, O. J..

10 recordsLinked to original sources

bioRxiv2026

Functional interplay between H2B ubiquitylation and H2A.Z deposition

The Bre1 ubiquitin conjugating enzyme catalyzes the monoubiquitylation of histone H2B-K120/K123 at promoter proximal nucleosomes, contributing to transcriptional regulation. These same nucleosomes are targeted by the yeast SWR1C chromatin remodeling enzyme that deposits the histone variant H2A.Z. Yeast strains that lack both Bre1 and Swr1 are inviable, indicating that together they contribute to an essential cell function. Interestingly, H2B-K123ub and H2A.Z levels are anticorrelated, and recent biochemical studies suggest a model in which H2B-K123ub inhibits SWR1C activity by blocking access to the nucleosome acidic patch. Here, we exploit recombinant nucleosomes to show that the H2A.Z deposition activity of SWR1C is strongly inhibited by H2B-K123ub. Surprisingly, the loss of H2B-K123ub does not lead to significant changes in the genomic organization of H2A.Z in cells grown in normal media, but we find that H2B-K123ub is required for a re-localization of H2A.Z from promoters to coding regions during replication stress. Together, these data indicate a complex functional interplay between H2B ubiquitylation and H2A.Z deposition.

biochemistry↗

bioRxiv2022

RNA polymerase II and PARP1 shape enhancer-promoter contacts

How enhancers control target gene expression over long genomic distances remains an important unsolved problem. Here we studied enhancer-promoter contact architecture and communication by integrating data from nucleosome-resolution genomic contact maps, nascent transcription, and perturbations to transcription-associated proteins and thousands of candidate enhancers. Contact frequency between functionally validated enhancer-promoter pairs was most enriched near the +1 and +2 nucleosomes at enhancers and target promoters, indicating that functional enhancer-promoter pairs spend time in close physical proximity. Blocking RNA polymerase II (Pol II) caused major disruptions to enhancer-promoter contacts. Paused Pol II occupancy and the enzymatic activity of poly (ADP-ribose) polymerase 1 (PARP1) stabilized enhancer-promoter contacts. Based on our findings, we propose an updated model that couples transcriptional dynamics and enhancer-promoter communication.

genomics↗

bioRxiv2019

Resolving the 3D landscape of transcription-linked mammalian chromatin folding

Chromatin folding below the scale of topologically associating domains (TADs) remains largely unexplored in mammals. Here, we used a high-resolution 3C-based method, Micro-C, to probe links between 3D-genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregate TAD regions into \"microTADs\" with distinct regulatory features. Enhancer-promoter and promoter-promoter interactions extending from the edge of these domains predominantly link co-regulated loci, often independently of CTCF/Cohesin. Acute inhibition of transcription disrupts the gene-related folding features without altering higher-order chromatin structures. Intriguingly, we detect \"two-start\" zig-zag 30-nanometer chromatin fibers. Our work uncovers the finer-scale genome organization that establishes novel functional links between chromatin folding and gene regulation.\n\nONE SENTENCE SUMMARYTranscriptional regulatory elements shape 3D genome architecture of microTADs.

genomics↗

bioRxiv2018

Control of noncoding RNA production and histone levels by a 5’ tRNA fragment

Small RNAs derived from mature tRNAs, referred to as tRNA fragments or \"tRFs\", are an emerging class of regulatory RNAs with poorly understood functions in cellular regulation. We recently identified a role for one specific tRF - 5 tRF-Gly-GCC, or tRFGG - in repression of genes associated with the endogenous retroelement MERVL, but the mechanistic basis for this regulation was unknown. Here, we show that tRF-GG plays a role in production of a wide variety of noncoding RNAs normally synthesized in Cajal bodies. Among these noncoding RNAs, tRF-GG regulation of the U7 snRNA modulates heterochromatin-mediated transcriptional repression of MERVL elements by supporting an adequate supply of histone proteins. Importantly, the effects of inhibiting tRF-GG on histone mRNA levels, activity of a histone 3 UTR reporter, and ultimately on MERVL regulation could all be suppressed by the U7 RNA. We show that the related RNA-binding proteins hnRNPF and H bind directly to tRF-GG, and are required for Cajal body biogenesis. Together, our data reveal a conserved mechanism for 5 tRNA fragment control of noncoding RNA biogenesis and, consequently, in global chromatin organization.

molecular biology↗

bioRxiv2018

Transitions in paternal dominance regulate offspring growth and metabolic transcription

Parental effects are an important source of adaptive traits. By contrast, parental effects failing to regulate offspring phenotype to fit current conditions could be deleterious. Although adaptive parental responses to single cues have been identified, we lack an understanding of the reversibility of parental effects across breeding cycles in a fluctuating environment. Social status of parents can occasionally fluctuate and, in turn, influence high-fitness pathways available to offspring. We show that social competition status results in robust parental effects on growth in mice. Dominant males produce faster growing offspring because of status related cues, not genetic associations. The timing, effect-size, and sex-specificity of this paternal effect are modulated by maternal experience. We experimentally demonstrate that status-ascending males produce heavier sons than before, and status-descending males produce lighter sons than before. Paternal status predicts genome-wide transcription in the liver, including transcriptional networks controlling xenobiotic and fatty acid metabolism, and oxidative phosphorylation. Our study demonstrates that paternal social status reversibly conditions offspring growth in naturalistic environments.

evolutionary biology↗

bioRxiv2018

Transfer RNA Genes Affect Chromosome Structure and Function via Local Effects

The genome is packaged and organized in an ordered, non-random manner and specific chromatin segments contact nuclear substructures to mediate this organization. While transfer RNA genes (tDNAs) are essential for the generation of tRNAs, these loci are also binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the role of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacks any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromosome folding or chromosome tethering. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long range HML-HMR heterochromatin clustering. We propose that the tDNAs primarily affect local chromatin structure that result in effects on long-range chromosome architecture.

molecular biology↗

bioRxiv2018

Condensin-dependent chromatin condensation represses transcription globally during quiescence

Quiescence is a stress-resistant state in which cells reversibly exit the mitotic cell cycle and suspend most cellular processes. Quiescence is essential for stem cell maintenance and its misregulation is implicated in tumor formation. One of the conserved hallmarks of quiescent cells, from Saccharomyces cerevisiae to humans, is highly condensed chromatin. Here, we use Micro-C XL to map chromatin contacts at single-nucleosome resolution genome-wide to elucidate mechanisms and functions of condensed chromatin in quiescent S. cerevisiae cells. We describe previously uncharacterized chromatin domains on the order of 10-60 kilobases that in quiescent cells are formed by condensin-mediated chromatin loops. Conditional depletion of condensin prevents chromatin condensation during quiescence entry and leads to widespread transcriptional de-repression. We further demonstrate that condensin-dependent chromatin compaction is conserved in quiescent human fibroblasts. We propose that condensin-dependent condensation of chromatin represses transcription throughout the quiescent cell genome.

molecular biology↗

bioRxiv2018

Profiling of pluripotency factors in individual stem cells and early embryos

Major cell fate decisions are governed by sequence-specific transcription factors (TFs) that act in small cell populations within developing embryos. To understand how TFs regulate cell fate it is important to identify their genomic binding sites in these populations. However, current methods cannot profile TFs genome-wide at or near the single cell level. Here we adapt the CUT&RUN method to profile chromatin proteins in low cell numbers, mapping TF-DNA interactions in single cells and individual pre-implantation embryos for the first time. Using this method, we demonstrate that the pluripotency TF NANOG is significantly more dependent on the SWI/SNF family ATPase BRG1 for association with its genomic targets in vivo than in cultured cells--a finding that could not have been made using traditional approaches. Ultra-low input CUT&RUN (uliCUT&RUN) enables interrogation of TF binding from low cell numbers, with broad applicability to rare cell populations of importance in development or disease.

genomics↗

bioRxiv2018

Effects of larval density on gene regulation in C. elegans during routine L1 synchronization

Bleaching gravid C. elegans followed by a short period of starvation of the L1 larvae is a routine method performed by worm researchers for generating synchronous populations for experiments. During the process of investigating dietary effects on gene regulation in L1 stage worms by single-worm RNA-Seq, we found that the density of resuspended L1 larvae affects expression of many mRNAs. Specifically, a number of genes related to metabolism and signalling are highly expressed in worms arrested at low density, but are repressed at higher arrest densities. We generated a GFP reporter strain based on one of the most density-dependent genes in our dataset - lips-15 - and confirmed that this reporter was expressed specifically in worms arrested at relatively low density. Finally, we show that conditioned media from high density L1 cultures was able to downregulate lips-15 even in L1 animals arrested at low density, and experiments using daf-22 mutant animals demonstrated that this effect is not mediated by the ascaroside family of signalling pheromones. Together, our data implicate a soluble signalling molecule in density sensing by L1 stage C. elegans, and provide guidance for design of experiments focused on early developmental gene regulation.

developmental biology↗

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↗