Biology subjects

Krebs, S.

Publications and source records attributed to Krebs, S..

26 records · Page 2Linked to original sources

bioRxiv2020

Human ORC/MCM density is low in active genes and correlates with replication time but does not solely define replication initiation zones

Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing and fork directionality profiles obtained by RNA-seq, Repli-seq and OK-seq. ORC and MCM are strongly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early-than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms that specify when and where replication initiates in human cells.

cell biology↗

bioRxiv2019

HIV-1 Envelope and MPER antibody structures in lipid assemblies

Structural and functional studies of HIV Env as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Thus, most structural studies have utilized soluble forms where the regions C-terminal to the ectodomain are deleted. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstituted a full-length Env clone into a nanodisc with MSP1D1 scaffold, complexed it with an MPER targeting antibody 10E8, and structurally defined the full quaternary epitope of 10E8 consisting of lipid, MPER and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapted the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with peptidisc scaffold.

immunology↗

bioRxiv2019

Active transcription regulates ORC/MCM distribution whereas replication timing correlates with ORC density in human cells

Eukaryotic replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with replication initiation events obtained by Okazaki fragment sequencing. We demonstrate that MCM is displaced from early replicating, actively transcribed gene bodies, while ORC is mainly enriched at active TSS. Late replicating, H4K20me3 containing initiation zones display enhanced ORC and MCM levels. Furthermore, we find early RTDs being primarily enriched in ORC, compared to MCM, indicating that ORC levels are involved in organizing the temporal order of DNA replication. The organizational connection between active transcription and replication competence directly links changes in the transcriptional program to flexible replication patterns, which ensures the cells flexibility to respond to environmental cues.

cell biology↗

bioRxiv2019

Epstein-Barr virus inactivates the transcriptome and disrupts the chromatin architecture of its host cell in the first phase of lytic reactivation

Epstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable long-term latent infection in human B cells, its preferred host. Upon induction of EBVs lytic phase the latently infected cells turn into a virus factory, a process, that is governed by EBV. In the lytic, productive phase all herpesviruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >105 binding sites with different sequence motifs in cellular chromatin and in a concentration dependent manner. Concomitant with DNA binding, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBVs lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficient de novo synthesis of this herpesvirus.

cell biology↗

bioRxiv2018

Antigen-specific T-cell receptor signatures of cytomegalovirus infection

Cytomegalovirus (CMV) is a prevalent human pathogen. The virus cannot be eliminated from the body, but is kept in check by CMV-specific T cells. Patients with an insufficient T-cell response, such as transplant recipients, are at high risk of developing CMV disease. However, the CMV-specific T-cell repertoire is complex, and is not yet clear which T cells protect best against virus reactivation and disease. Here we present a highly resolved characterization of CMV-specific CD8+ T cells based on enrichment by specific peptide stimulation and mRNA sequencing of their T-cell receptor {beta} chains (TCR{beta}). Our analysis included recently identified T-cell epitopes restricted through HLA-C, whose presentation is resistant to viral immunomodulation, and well-studied HLA-B-restricted epitopes. In 8 healthy virus carriers, we identified a total of 1052 CMV-specific TCR{beta} chains. HLA-C-restricted, CMV-specific TCR{beta} clonotypes the ex vivo T-cell response, and contributed the highest-frequency clonotype of the entire repertoire in 2 of 8 donors. We analyzed sharing and similarity of CMV-specific TCR{beta} sequences and identified 63 public or related sequences belonging to 17 public TCR{beta} families. In our cohort and in an independent cohort of 352 donors, the cumulative frequency of these public TCR{beta} family members was a highly discriminatory indicator of carrying both CMV infection and the relevant HLA type. Based on these findings, we propose CMV-specific TCR{beta} signatures as a biomarker for an antiviral T-cell response to identify patients in need of treatment and to guide future development of immunotherapy.

immunology↗

bioRxiv2018

Genome-wide measurement of local nucleosome array regularity and spacing by nanopore sequencing

The nature of chromatin as regular succession of nucleosomes has gained iconic status. However, since most nucleosomes in metazoans are poorly positioned it is unknown to which extent the bulk genomic nucleosome repeat length (NRL) reflects the regularity and spacing of nucleosome arrays at individual loci. We describe a new approach to map nucleosome array regularity and spacing through sequencing oligonucleosome-derived DNA by Illumina sequencing as well as emergent nanopore-technology. This revealed modulation of array regularity and NRL depending on functional chromatin states independently of nucleosome phasing and even in unmappable regions. We also found that nucleosome arrays downstream of silent promoters are considerably more regular than those downstream of highly expressed ones, despite more extensive nucleosome phasing of the latter. Our approach is generally applicable and provides an important parameter of chromatin organisation that so far had been missing.

genomics↗

bioRxiv2017

A taxogenomics approach uncovers a new genus in the phylum Placozoa

The Placozoa [1] is a monotypic phylum of non-bilaterian marine animals. Its only species, Trichoplax adhaerens, was described in 1883 [2], Despite the worldwide distribution of placozoans [3-6], morphological differences are lacking among isolates from different geographic areas and, consequently, no other species in this phylum has been described and accepted for more than 130 years. However, recent single-gene studies on the genetic diversity of this \"species\" have revealed deeply divergent lineages of, as yet, undefined taxonomic ranks [3,5,6], Since single genes are not considered sufficient to define species [7], a whole nuclear genome comparison appears the most appropriate approach to determine relationships between placozoan lineages. Such a \"taxogenomics\" approach can help discover and diagnose potential additional species and, therefore, develop a much-needed, more robust, taxonomic framework for this phylum. To achieve this we sequenced the genome of a placozoan lineage isolated from Hong Kong (lineage H13), which is distantly related to T. adhaerens [6]. The 87 megabase genome assembly contains 12,010 genes. Comparison to the T. adhaerens genome [8] identified an average protein distance of 24.4% in more than 2,700 screened one-to-one orthologs, similar to levels observed between the chordate classes mammals and birds. Genome rearrangements are commonplace and >25% of genes are not collinear (i.e. they are not in the same order in the two genomes). Finally, a multi-gene distance comparison with other non-bilaterian phyla indicate genus level differences to T. adhaerens. These data highlight the large genomic diversity within the Placozoa and justifies the designation of lineage HI3 as a new species, Xxxxxxxxx yyyyyyyyyyyyy1 gen. et spaec. nov., now the second described placozoan species and the first in a new genus. Phylogenomic analyses furthermore supports a robust placement of the Placozoa as sister to a cnidarian-bilaterian clade.

evolutionary biology↗

bioRxiv2017

The Genome Of The Contractile Demosponge Tethya wilhelma And The Evolution Of Metazoan Neural Signalling Pathways

Porifera are a diverse animal phylum with species performing important ecological roles in aquatic ecosystems, and have become models for multicellularity and early-animal evolution. Demosponges form the largest class in sponges, but previous studies have relied on the only draft demosponge genome of Amphimedon queenslandica. Here we present the 125-megabase draft genome of a contractile laboratory demosponge Tethya wilhelma, sequenced to almost 150x coverage. We explore the genetic repertoire of transporters, receptors, and neurotransmitter metabolism across early-branching metazoans in the context of the evolution of these gene families. Presence of many genes is highly variable across animal groups, with many gene family expansions and losses. Three sponge classes show lineage-specific expansions of GABA-B receptors, far exceeding the gene number in vertebrates, while ctenophores appear to have secondarily lost most genes in the GABA pathway. Both GABA and glutamate receptors show lineage-specific domain rearrangements, making it difficult to trace the evolution of these gene families. Gene sets in the examined taxa suggest that nervous systems evolved independently at least twice and either changed function or were lost in sponges. Changes in gene content are consistent with the view that ctenophores and sponges are the earliest-branching metazoan lineages and provide additional support for the proposed clade of Placozoa/Cnidaria/Bilateria.

genomics↗