Biology subjects

Lee, M. T.

Publications and source records attributed to Lee, M. T..

10 recordsLinked to original sources

bioRxiv2024

H3K4me2 distinguishes a distinct class of enhancers during the maternal-to-zygotic transition

After egg fertilization, an initially silent embryonic genome is transcriptionally activated during the maternal-to-zygotic transition. In zebrafish, maternal vertebrate pluripotency factors Nanog, Pou5f3 (OCT4 homolog), and Sox19b (SOX2 homolog) (NPS) play essential roles in orchestrating embryonic genome activation, acting as "pioneers" that open condensed chromatin and mediate acquisition of activating histone modifications. However, some embryonic gene transcription still occurs in the absence of these factors, suggesting the existence of other mechanisms regulating genome activation. To identify chromatin signatures of these unknown pathways, we profiled the histone modification landscape of zebrafish embryos using CUT&RUN. Our regulatory map revealed two subclasses of enhancers distinguished by presence or absence of H3K4me2. Enhancers lacking H3K4me2 tend to require NPS factors for de novo activation, while enhancers bearing H3K4me2 are epigenetically bookmarked by DNA hypomethylation to recapitulate gamete activity in the embryo, independent of NPS pioneering. Thus, parallel enhancer activation pathways combine to induce transcriptional reprogramming to pluripotency in the early embryo.

developmental biology↗

bioRxiv2023

Parallels and contrasts between the cnidarian and bilaterian maternal-to-zygotic transition are revealed in Hydractinia embryos

Embryogenesis requires coordinated gene regulatory activities early on that establish the trajectory of subsequent development, during a period called the maternal-to-zygotic transition (MZT). The MZT comprises transcriptional activation of the embryonic genome and post-transcriptional regulation of egg-inherited maternal mRNA. Investigation into the MZT in animals has focused almost exclusively on bilaterians, which include all classical models such as flies, worms, sea urchin, and vertebrates, thus limiting our capacity to understand the gene regulatory paradigms uniting the MZT across all animals. Here, we elucidate the MZT of a non-bilaterian, the cnidarian Hydractinia symbiolongicarpus. Using parallel poly(A)-selected and non poly(A)-dependent RNA-seq approaches, we find that the Hydractinia MZT is composed of regulatory activities analogous to many bilaterians, including cytoplasmic readenylation of maternally contributed mRNA, delayed genome activation, and separate phases of maternal mRNA deadenylation and degradation that likely depend on both maternally and zygotically encoded clearance factors, including microRNAs. But we also observe massive upregulation of histone genes and an expanded repertoire of predicted H4K20 methyltransferases, aspects thus far unique to the Hydractinia MZT and potentially underlying a novel mode of early embryonic chromatin regulation. Thus, similar regulatory strategies with taxon-specific elaboration underlie the MZT in both bilaterian and non-bilaterian embryos, providing insight into how an essential developmental transition may have arisen in ancestral animals.

developmental biology↗

bioRxiv2022

Hybridization led to a rewired pluripotency network in the allotetraploid Xenopus laevis

After fertilization, maternally contributed factors to the egg initiate the transition to pluripotency to give rise to embryonic stem cells, in large part by activating de novo transcription from the embryonic genome. Diverse mechanisms coordinate this transition across animals, suggesting that pervasive regulatory remodeling has shaped the earliest stages of development. Here, we show that maternal homologs of mammalian pluripotency reprogramming factors OCT4 and SOX2 divergently activate the two subgenomes of Xenopus laevis, an allotetraploid that arose from hybridization of two diploid species ~18 million years ago. Although most genes have been retained as two homeologous copies, we find that a majority of them undergo asymmetric activation in the early embryo. Chromatin accessibility profiling and CUT&RUN for modified histones and transcription factor binding reveal extensive differences in enhancer architecture between the subgenomes, which likely arose through genomic disruptions as a consequence of allotetraploidy. However, comparison with diploid X. tropicalis and zebrafish shows broad conservation of embryonic gene expression levels when divergent homeolog contributions are combined, implying strong selection to maintain dosage in the core vertebrate pluripotency transcriptional program, amid genomic instability following hybridization.

developmental biology↗

bioRxiv2021

Positive modulation of cerebellar α6GABAA receptors for treating essential tremor: a proof-of-concept study in harmaline-treated mice

BackgroundThe etiology of essential tremor (ET) remains unclear but may involve abnormal firing of Purkinje cells, which receive excitatory inputs from granule cells in the cerebellum. Since 6 subunit-containing GABAA receptors (6GABAARs) are abundantly expressed in granule cells, we validated a hypothesis that 6GABAAR-selective positive allosteric modulators (PAMs) are promising pharmacological interventions for ET therapy. MethodsEmploying the harmaline-induced ET model in male ICR mice, we evaluated the possible anti-tremor effects of four 6GABAAR-selective PAMs, the pyrazoloquinolinones Compound 6 and LAU-463 and their respective deuterated derivatives. Propranolol, a clinical anti-tremor agent, was employed as positive control. To investigate the involvement of cerebellar 6GABAARs in the antitremor effect of intraperitoneal (i.p.) Compound 6, furosemide, an 6GABAAR antagonist, was intracerebellarly (i.cb.) co-administered with Compound 6. The burrowing activity, an indicator of wellbeing in rodents, was measured concurrently. ResultsHarmaline (10-30 mg/kg, s.c.) induced action tremor in ICR mice dose-dependently and markedly reduced their burrowing activity. Compound 6 (3 and 10 mg/kg, i.p.) significantly attenuated harmaline (20 mg/kg)-induced action tremor and burrowing activity impairment. Propranolol (20 mg/kg, i.p.) diminished tremor but failed to restore the burrowing activity in harmaline-treated mice. Importantly, both anti-tremor and burrowing activity restorative effects of Compound 6 (10 mg/kg, i.p.) was significantly reversed by co-administration of i.cb. furosemide at a dose (10 nmol/0.5 l) having no effect per se. All four 6GABAAR PAMs exhibited a similar therapeutic efficacy. Conclusion6GABAAR-selective PAMs significantly attenuated action tremor and restored physical well-being in a mouse model mimicking ET by acting in the cerebellum. Thus, 6GABAAR-selective PAMs may be potential therapeutic agents for ET.

pharmacology and toxicology↗

bioRxiv2020

RNA degradation sculpts the maternal transcriptome during Drosophila oogenesis

In sexually reproducing animals, the oocyte contributes a large supply of RNAs that are essential to launch development upon fertilization. The mechanisms that regulate the composition of the maternal RNA contribution during oogenesis are unclear. Here, we show that a subset of RNAs expressed during the early stages of oogenesis is subjected to regulated degradation during oocyte specification. Failure to remove these RNAs results in oocyte dysfunction and death. We identify the RNA-degrading Super Killer complex and No-Go Decay factor Pelota as key regulators of oogenesis via targeted clearance of RNAs expressed in germline stem cells. These regulators target RNAs enriched for cytidine sequences bound by the protein Half pint. Thus, RNA degradation helps orchestrate a germ cell-to-maternal transition by sculpting the maternal RNA contribution to the zygote.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2020

Optimized design of antisense oligomers for targeted rRNA depletion

RNA sequencing (RNA-seq) has become a standard method for quantifying gene expression transcriptome-wide. Although RNA-seq is often paired with polyadenylate (poly(A)) selection to enrich for messenger RNA (mRNA), many applications require alternate approaches to counteract the high proportion of ribosomal RNA (rRNA) in total RNA. Recently, selective rRNA digestion, using RNaseH and antisense DNA oligomers that tile the entire length of target RNAs, has emerged as an alternative to commercial rRNA depletion kits. Here, we present a streamlined, more economical RNaseH-mediated rRNA depletion method with substantially lower up-front costs, using shorter antisense oligos only sparsely tiled along the target RNA, in a digestion reaction of only 5 minutes. We introduce a novel Web tool, Oligo-ASST, that simplifies oligo design to target regions with optimal thermodynamic properties, and additionally can generate compact, common oligo pools that simultaneously target divergent RNAs, e.g. across different species. We demonstrate the efficacy of these strategies by designing oligo sets to deplete rRNA in Xenopus laevis and in zebrafish, which expresses two distinct versions of rRNAs during embryogenesis. The resulting RNA-seq libraries reduce rRNA to <5% of aligned reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available at https://mtleelab.pitt.edu/oligo to design antisense oligos for any taxon or to target any abundant RNA for depletion. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/169102v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@c05b57org.highwire.dtl.DTLVardef@ace8a3org.highwire.dtl.DTLVardef@1dd25adorg.highwire.dtl.DTLVardef@1d4adeb_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Zinc protection of fertilized eggs is an ancient feature of sexual reproduction in animals

One of the earliest and most prevalent barriers to successful reproduction is polyspermy, or fertilization of an egg by multiple sperm. To prevent these supernumerary fertilizations, eggs have evolved multiple mechanisms. It has recently been proposed that zinc released by mammalian eggs at fertilization may block additional sperm from entering. Here, we demonstrate that eggs from amphibia and teleost fish also release zinc. Using Xenopus laevis as a model, we document that zinc reversibly blocks fertilization. Finally, we demonstrate that extracellular zinc similarly disrupts early embryonic development in eggs from diverse phyla, including: Cnidaria, Echinodermata, and Chordata. Our study reveals that a fundamental strategy protecting human eggs from fertilization by multiple sperm may have evolved more than 650 million years ago.Competing Interest StatementThe authors have declared no competing interest.View Full Text

physiology↗

bioRxiv2019

The Paf1 complex broadly impacts the transcriptome of Saccharomyces cerevisiae

The Polymerase Associated Factor 1 complex (Paf1C) is a multifunctional regulator of eukaryotic gene expression important for the coordination of transcription with chromatin modification and post-transcriptional processes. In this study, we investigated the extent to which the functions of Paf1C combine to regulate the Saccharomyces cerevisiae transcriptome. While previous studies focused on the roles of Paf1C in controlling mRNA levels, here we took advantage of a genetic background that enriches for unstable transcripts and demonstrate that deletion of PAF1 affects all classes of Pol II transcripts including multiple classes of noncoding RNAs. By conducting a de novo differential expression analysis independent of gene annotations, we found that Paf1 positively and negatively regulates antisense transcription at multiple loci. Comparisons with nascent transcript data revealed that many, but not all, changes in RNA levels detected by our analysis are due to changes in transcription instead of post-transcriptional events. To investigate the mechanisms by which Paf1 regulates protein-coding genes, we focused on genes involved in iron and phosphate homeostasis, which were differentially affected by PAF1 deletion. Our results indicate that Paf1 stimulates phosphate gene expression through a mechanism that is independent of any individual Paf1C-dependent histone modification. In contrast, the inhibition of iron gene expression by Paf1 correlates with a defect in H3 K36 tri-methylation. Finally, we showed that one iron regulon gene, FET4, is coordinately controlled by Paf1 and transcription of upstream noncoding DNA. Together these data identify roles for Paf1C in controlling both coding and noncoding regions of the yeast genome.

genomics↗

bioRxiv2018

A post-transcriptional regulatory code for mRNA stability during the zebrafish maternal-to-zygotic transition

Post-transcriptional regulation is crucial to shape gene expression. During the Maternal-to-Zygotic Transition (MZT), thousands of maternal transcripts are regulated upon fertilization and genome activation. Transcript stability can be influenced by cis-elements and trans-factors, but how these inputs are integrated to determine the overall mRNA stability is unclear. Here, we show that most transcripts are under combinatorial regulation by multiple decay pathways during zebrafish MZT. To identify cis-regulatory elements, we performed a massively parallel reporter assay for stability-influencing sequences, which revealed that 3-UTR poly-U motifs are associated with mRNA stability. In contrast, miR-430 target sequences, UAUUUAUU AU-rich elements (ARE), CCUC and CUGC elements emerged as the main destabilizing motifs in the embryo, with miR-430 and AREs causing mRNA deadenylation in a genome activation-dependent manner. To identify the trans-factors interacting with these cis-elements, we comprehensively profiled RNA-protein interactions and their associated regulatory activities across the transcriptome during the MZT. We find that poly-U binding proteins are preferentially associated with 3-UTR sequences and stabilizing motifs, and that antagonistic sequence contexts for poly-C and poly-U binding proteins shape the binding landscape and magnitude of regulation across the transcriptome. Finally, we integrate these regulatory motifs into a machine learning model that accurately predicts the stability of mRNA reporters in vivo. Our findings reveal how mechanisms of post-transcriptional regulation are coordinated to direct changes in mRNA stability within the early zebrafish embryo.

genomics↗

bioRxiv2018

The TMEM16A Channel Mediates the fast polyspermy block in Xenopus Laevis

In externally fertilizing animals, such as sea urchins and frogs, prolonged depolarization of the egg immediately after fertilization inhibits the entry of additional sperm - a phenomenon known as the fast block to polyspermy. In the African clawed frog, Xenopus laevis, this depolarization is driven by a Ca2+-activated Cl- efflux. Although the prominent Ca2+-activated Cl- currents generated by immature X. laevis oocytes are conducted by xTMEM16A channels, little is known about which channels contribute to fertilization-competency in mature eggs. Moreover, the gamete undergoes a gross transformation as it matures from an immature oocyte into a fertilization-competent egg. Here we report the results of our approach to identify the Ca2+-activated Cl- channel that triggers the fast block. Querying published proteomics and RNA-seq data, we identified two Ca2+-activated Cl- channels expressed in fertilization-competent X. laevis eggs: xTMEM16A and xBEST2A. Furthermore, transcripts for these channels increase in abundance during gamete maturation. To determine if either of these mediates the fast block, we characterized exogenously expressed xTMEM16A and xBEST2A using pharmacologic inhibitors. None of the inhibitors tested blocked xBEST2A currents specifically. However, Ani9 and MONNA each reduced xTMEM16A currents by more than 70%, while only nominally inhibiting those generated by xBEST2A. Using whole-cell recordings during fertilization, we found that Ani9 and MONNA effectively diminished fertilization-evoked depolarizations. These results indicate that fertilization activates TMEM16A channels in X. laevis eggs and induces the earliest known event triggered by fertilization: the fast block to polyspermy.\n\nHIGHLIGHTSO_LIProtein for the channels xBEST2A and xTMEM16A is present in X. laevis eggs.\nC_LI\n\nO_LIThe inhibitors MONNA and Ani9 effectively block xTMEM16A compared to xBEST2A.\nC_LI\n\nO_LIXenopus laevis fertilization opens TMEM16A to trigger egg depolarization.\nC_LI\n\nO_LIThe TMEM16A-mediated depolarization is critical for the fast block to polyspermy.\nC_LI

developmental biology↗