Balancing transcriptional activity in Drosophila through protein-protein interactions on chromatin

Chromatin plays a vital role in the implementation of gene expression programs. Several disparate groups of regulatory proteins alter chromatin state through post-translational modification of histone proteins, nucleosome remodeling, and higher order chromatin structure in order to affect gene expression. Several of these key groups, such as the Male-Specific Lethal complex and Polycomb Group have been well characterized in Drosophila. Yet aspects of their biology at the molecular level, such as the means by which they are faithfully targeted to regulated loci throughout the genome and the molecular mechanisms they employ to alter transcriptional state, still remain unexplained. In this dissertation I explore how identifying protein-protein interactions on chromatin reveals insights into these unanswered questions critical to chromatin biology. My results highlight the importance of balancing active and repressive chromatin states for the proper maintenance of gene expression. The Male-Specific Lethal complex is the dosage compensation complex in Drosophila, which upregulates gene expression on the male X chromosome approximately two-fold. The MSL complex catalyzes an acetyl mark which may create a uniquely permissive chromatin state to promote transcriptional elongation. A proteomic screen for MSL-interacting proteins identified UpSET, the Drosophila homolog of yeast SET3 and mammalian MLL5. Interestingly, SET3 and UpSET have been characterized to assemble into histone deacetylase complexes. I employed genetic, genomic, and proteomic techniques to assess whether UpSET plays a role in dosage compensation. UpSET appears to play a role in limiting the level of activation of the MSL complex. Surprisingly, UpSET appears to play a more important role in the maintenance of heterochromatin. The Polycomb Group is comprised of a well characterized set of developmental repressors. The PcG assembles into several multiprotein complexes to maintain the repressed state. The PcG is opposed by a group of activators known as the Trithorax group. Although the PcG and TrxG often appear to be recruited to the same genomic elements in different tissues, whether they might interact directly was not known. In a collaboration with Dr. Hyuckjoon Kang, I characterized the TrxG protein Female sterile (1) homeotic and found that it interacts specifically with PRC1. The data support a model that bivalency, a poised state observed in mammalian stem cells, may be critical, perhaps transiently, in the developing Drosophila embryo. The mechanism of coordination amongst the various PcG complexes on chromatin is not well understood. We also identified the Sex comb on midleg protein, a known member of the PcG, as a potential physical bridge between PRC1 and PRC2. In these sets of experiments, I have characterized instances of crosstalk between activating and repressing regulators which are critical for the proper maintenance of chromatin state. Perturbations of these interactions may lead to an imbalance of regulators on chromatin and aberrant transcriptional activity. These findings highlight the need for tuning gene expression state and suggest chromatin-based mechanisms by which this can be accomplished.Biology, Molecular and Cellula

Are we there yet? Initial targeting of the Male-Specific Lethal and Polycomb group chromatin complexes in Drosophila

Chromatin-binding proteins must navigate the complex nuclear milieu to find their sites of action, and a constellation of protein factors and other properties are likely to influence targeting specificity. Despite considerable progress, the precise rules by which binding specificity is achieved have remained elusive. Here, we consider early targeting events for two groups of chromatin-binding complexes in Drosophila: the Male-Specific Lethal (MSL) and the Polycomb group (PcG) complexes. These two serve as models for understanding targeting, because they have been extensively studied and play vital roles in Drosophila, and their targets have been documented at high resolution. Furthermore, the proteins and biochemical properties of both complexes are largely conserved in multicellular organisms, including humans. While the MSL complex increases gene expression and PcG members repress genes, the two groups share many similarities such as the ability to modify their chromatin environment to create active or repressive domains, respectively. With legacies of in-depth genetic, biochemical and now genomic approaches, the MSL and PcG complexes will continue to provide tractable systems for understanding the recruitment of multiprotein chromatin complexes to their target loci

Opsin expression varies across larval development and taxa in pteriomorphian bivalves

IntroductionMany marine organisms have a biphasic life cycle that transitions between a swimming larva with a more sedentary adult form. At the end of the first phase, larvae must identify suitable sites to settle and undergo a dramatic morphological change. Environmental factors, including photic and chemical cues, appear to influence settlement, but the sensory receptors involved are largely unknown. We targeted the protein receptor, opsin, which belongs to large superfamily of transmembrane receptors that detects environmental stimuli, hormones, and neurotransmitters. While opsins are well-known for light-sensing, including vision, a growing number of studies have demonstrated light-independent functions. We therefore examined opsin expression in the Pteriomorphia, a large, diverse clade of marine bivalves, that includes commercially important species, such as oysters, mussels, and scallops.MethodsGenomic annotations combined with phylogenetic analysis show great variation of opsin abundance among pteriomorphian bivalves, including surprisingly high genomic abundance in many species that are eyeless as adults, such as mussels. Therefore, we investigated the diversity of opsin expression from the perspective of larval development. We collected opsin gene expression in four families of Pteriomorphia, across three distinct larval stages, i.e., trochophore, veliger, and pediveliger, and compared those to adult tissues.ResultsWe found larvae express all opsin types in these bivalves, but opsin expression patterns are largely species-specific across development. Few opsins are expressed in the adult mantle, but many are highly expressed in adult eyes. Intriguingly, opsin genes such as retinochrome, xenopsins, and Go-opsins have higher levels of expression in the later larval stages when substrates for settlement are being tested, such as the pediveliger.ConclusionInvestigating opsin gene expression during larval development provides crucial insights into their intricate interactions with the surroundings, which may shed light on how opsin receptors of these organisms respond to various environmental cues that play a pivotal role in their settlement process

Lineage Abundance Estimation for SARS-CoV-2 in Wastewater Using Transcriptome Quantification Techniques

Effectively monitoring the spread of SARS-CoV-2 mutants is essential to efforts to counter the ongoing pandemic. Predicting lineage abundance from wastewater, however, is technically challenging. We show that by sequencing SARS-CoV-2 RNA in wastewater and applying algorithms initially used for transcriptome quantification, we can estimate lineage abundance in wastewater samples. We find high variability in signal among individual samples, but the overall trends match those observed from sequencing clinical samples. Thus, while clinical sequencing remains a more sensitive technique for population surveillance, wastewater sequencing can be used to monitor trends in mutant prevalence in situations where clinical sequencing is unavailable

Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders

Genetic influences on psychiatric disorders transcend diagnostic boundaries, suggesting substantial pleiotropy of contributing loci. However, the nature and mechanisms of these pleiotropic effects remain unclear. We performed analyses of 232,964 cases and 494,162 controls from genome-wide studies of anorexia nervosa, attention-deficit/hyper-activity disorder, autism spectrum disorder, bipolar disorder, major depression, obsessive-compulsive disorder, schizophrenia, and Tourette syndrome. Genetic correlation analyses revealed a meaningful structure within the eight disorders, identifying three groups of inter-related disorders. Meta-analysis across these eight disorders detected 109 loci associated with at least two psychiatric disorders, including 23 loci with pleiotropic effects on four or more disorders and 11 loci with antagonistic effects on multiple disorders. The pleiotropic loci are located within genes that show heightened expression in the brain throughout the lifespan, beginning prenatally in the second trimester, and play prominent roles in neurodevelopmental processes. These findings have important implications for psychiatric nosology, drug development, and risk prediction.Peer reviewe

Molluscan Genomes Reveal Extensive Differences in Photopigment Evolution Across the Phylum

In animals, opsins and cryptochromes are major protein families that transduce light signals when bound to light-absorbing chromophores. Opsins are involved in various light-dependent processes, like vision, and have been co-opted for light-independent sensory modalities. Cryptochromes are important photoreceptors in animals, generally regulating circadian rhythm, they belong to a larger protein family with photolyases, which repair UV-induced DNA damage. Mollusks are great animals to explore questions about light sensing as eyes have evolved multiple times across, and within, taxonomic classes. We used molluscan genome assemblies from 80 species to predict protein sequences and examine gene family evolution using phylogenetic approaches. We found extensive opsin family expansion and contraction, particularly in bivalve xenopsins and gastropod Go-opsins, while other opsins, like retinochrome, rarely duplicate. Bivalve and gastropod lineages exhibit fluctuations in opsin repertoire, with cephalopods having the fewest number of opsins and loss of at least 2 major opsin types. Interestingly, opsin expansions are not limited to eyed species, and the highest opsin content was seen in eyeless bivalves. The dynamic nature of opsin evolution is quite contrary to the general lack of diversification in mollusk cryptochromes, though some taxa, including cephalopods and terrestrial gastropods, have reduced repertoires of both protein families. We also found complete loss of opsins and cryptochromes in multiple, but not all, deep-sea species. These results help set the stage for connecting genomic changes, including opsin family expansion and contraction, with differences in environmental, and biological features across Mollusca.This article is published as Kyle E McElroy, Jorge A Audino, Jeanne M Serb, Molluscan Genomes Reveal Extensive Differences in Photopigment Evolution Across the Phylum, Molecular Biology and Evolution, Volume 40, Issue 12, December 2023, msad263, https://doi.org/10.1093/molbev/msad263. © The Author(s) 2023.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited

Chromatin modification by PSC occurs at one PSC per nucleosome and does not require the acidic patch of histone H2A.

Chromatin architecture is regulated through both enzymatic and non-enzymatic activities. For example, the Polycomb Group (PcG) proteins maintain developmental gene silencing using an array of chromatin-based mechanisms. The essential Drosophila PcG protein, Posterior Sex Combs (PSC), compacts chromatin and inhibits chromatin remodeling and transcription through a non-enzymatic mechanism involving nucleosome bridging. Nucleosome bridging is achieved through a combination of nucleosome binding and self-interaction. Precisely how PSC interacts with chromatin to bridge nucleosomes is not known and is the subject of this work. We determine the stoichiometry of PSC-chromatin interactions in compact chromatin (in which nucleosomes are bridged) using Scanning Transmission Electron Microscopy (STEM). We find that full compaction occurs with one PSC per nucleosome. In addition to compacting chromatin, we show that PSC oligomerizes nucleosome arrays. PSC-mediated oligomerization of chromatin occurs at similar stoichiometry as compaction suggesting it may also involve nucleosome bridging. Interactions between the tail of histone H4 and the acidic patch of histone H2A are important for chromatin folding and oligomerization, and several chromatin proteins bind the histone H2A acidic patch. However, mutation of the acidic patch of histone H2A does not affect PSC's ability to inhibit chromatin remodeling or bridge nucleosomes. In fact, PSC does not require nucleosomes for bridging activity but can bridge naked DNA segments. PSC clusters nucleosomes on sparsely assembled templates, suggesting it interacts preferentially with nucleosomes over bare DNA. This may be due to the ability of PSC to bind free histones. Our data are consistent with a model in which each PSC binds a nucleosome and at least one other PSC to directly bridge nucleosomes and compact chromatin, but also suggest that naked DNA can be included in compacted structures. We discuss how our data highlight the diversity of mechanisms used to modify chromatin architecture

Summary of Measurements of Electron Micrographs of Chromatin with PSC.

<p>Table indicates mean +/− standard deviation for three measurements from two experiments. Each template uses a plasmid containing two 601 nucleosome positioning sequences that is assembled at low ratios of histones to DNA (0.2 histone:DNA by mass). The first set template has ∼1600 bp between the 601 nucleosomes and the second has 385 bp. Diameter is the maximum diameter of each template (the diameter of the smallest circle that would completely encompass the template) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047162#pone.0047162-Francis1" target="_blank">[15]</a>. The maximal diameter of each particle on each template was measured, giving rise to the “particle diameter” measurement. For –PSC samples, particles should be nucleosomes, while in +PSC samples, they could be nucleosomes, PSC bound to naked DNA, or PSC bound nucleosomes. Note that the largest diameter of the disk-shaped nucleosome is 11 nm; samples were rotary shadowed to a thickness of 3.75 nm; thus, the diameter measured is consistent with expectation (11+2×3.75 = 18.5 nm predicted size). The number of particles indicates each separate particle on a template, irrespective of size.</p

PSC does not require histone modifications or the acidic patch of H2A to inhibit chromatin remodeling.

<p>(a) Amino acid sequences for the wild-type H2A acidic patch (WT) and uncharged mutant (STT). Acidic residues are highlighted, and mutated residues are underlined. (b) MgCl₂ dependent oligomerization of wild type and H2A-STT containing chromatin. Chromatin was incubated with the indicated concentrations of MgCl₂ and centrifuged in a microfuge. Supernatants were electrophoresed on agarose gels and stained with SYBR gold; the % of the template remaining in the supernatant was determined by comparison with the 0 mM MgCl₂ control. (c) Summary of chromatin oligomerization assays. (d) Restriction enzyme accessibility (REA) assays on chromatin templates with 12 nucleosomes. The chromatin template contains a unique restriction site (HhaI) that is normally occluded by nucleosomes but is exposed upon Swi/Snf-mediated chromatin remodeling. The first two lanes are negative and positive controls (with or without Swi/Snf, no PSC) demonstrating that the HhaI site becomes more accessible in the presence of Swi/Snf. (e) Summary of REA assay on chromatin templates assembled with rec-WT and H2A-STT histones. Percent inhibition is calculated as .</p

PSC compacts chromatin at a ratio of 1∶1 with nucleosomes.

<p>(a) Representative EM images of negatively stained PSC. (b) Distribution of diameters of negatively stained PSC (n = 235). (c) Mass distributions of STEM analysis of PSC alone (n = 515). (d) Summary of measured masses of PSC. (e) Mass distributions of STEM analyses. Measured masses for 4N and 4N + PSC are 0.97±0.01 MDa (n = 130) and 1.67±0.03 MDa (n = 86) respectively.</p