Large-scale analyses of human microbiomes reveal thousands of small, novel genes and their predicted functions, bioRxiv, 2018-12-14
AbstractSmall proteins likely abound in prokaryotes, and may mediate much of the communication that occurs between organisms within a microbiome and their host. Unfortunately, small proteins are traditionally overlooked in biology, in part due to the computational and experimental difficulties in detecting them. To systematically identify novel small proteins, we carried out a large comparative genomics study on 1,773 HMP human-associated metagenomes from four different body sites (mouth, gut, skin and vagina). We describe more than four thousand conserved protein families, the majority of which are novel; ~30% of these protein families are predicted to be secreted or transmembrane. Over 90% of the small protein families have no known domain, and almost half are not represented in reference genomes, emphasizing the incompleteness of knowledge in this space. Our analysis exposes putative novel ‘housekeeping’ small protein families, including a potential novel ribosomally associated protein, as well as ‘mammalian-specific’ or ‘human-specific’ protein families. By analyzing the genomic neighborhood of small genes, we pinpoint a subset of families that are potentially associated with defense against bacteriophage. Finally, we identify families that may be subject to horizontal transfer and are thus potentially involved in adaptation of bacteria to the changing human environment. Our study suggest that small proteins are highly abundant and that those of the human microbiome, in particular, may perform diverse functions that have not been previously reported.
biorxiv microbiology 0-100-users 2018Population structure of modern-day Italians reveals patterns of ancient and archaic ancestries in Southern Europe, bioRxiv, 2018-12-14
European populations display low genetic diversity as the result of long term blending of the small number of ancient founding ancestries. However it is still unclear how the combination of ancient ancestries related to early European foragers, Neolithic farmers and Bronze Age nomadic pastoralists can fully explain genetic variation across Europe. Populations in natural crossroads like the Italian peninsula are expected to recapitulate the overall continental diversity, but to date have been systematically understudied. Here we characterised the ancestry profiles of modern-day Italian populations using a genome-wide dataset representative of modern and ancient samples from across Italy, Europe and the rest of the world. Italian genomes captured several ancient signatures, including a non-steppe related substantial ancestry contribution ultimately from the Caucasus. Differences in ancestry composition as the result of migration and admixture generated in Italy the largest degree of population structure detected so far in the continent and shaped the amount of Neanderthal DNA present in modern-day populations.
biorxiv genomics 0-100-users 2018An RNA-binding region regulates CTCF clustering and chromatin looping, bioRxiv, 2018-12-13
Mammalian genomes are folded into Topologically Associating Domains (TADs), consisting of cell-type specific chromatin loops anchored by CTCF and cohesin. Since CTCF and cohesin are expressed ubiquitously, how cell-type specific CTCF-mediated loops are formed poses a paradox. Here we show RNase-sensitive CTCF self-association in vitro and that an RNA-binding region (RBR) mediates CTCF clustering in vivo. Intriguingly, deleting the RBR abolishes or impairs almost half of all chromatin loops in mouse embryonic stem cells. Disrupted loop formation correlates with abrogated clustering and diminished chromatin binding of the RBR mutant CTCF protein, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least 2 classes RBR-independent and RBR-dependent loops. We suggest that evidence for distinct classes of RBR-dependent loops may provide a mechanism for establishing cell-specific CTCF loops regulated by RNAs and other RBR partner.
biorxiv biophysics 200-500-users 2018Comprehensive Immune Monitoring of Clinical Trials to Advance Human Immunotherapy, bioRxiv, 2018-12-13
SummaryThe success of immunotherapy has led to a myriad of new clinical trials. Connected to these trials are efforts to discover biomarkers providing mechanistic insight and predictive signatures for personalization. Still, the plethora of immune monitoring technologies can face investigator bias, missing unanticipated cellular responses in limited clinical material. We here present a mass cytometry workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate human immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. The resulting assay enumerated ≥ 98% of peripheral immune cells with ≥ 4 positively identifying antigens. Robustness and reproducibility were demonstrated on multiple samples types, across research centers and by orthogonal measurements. Using automated analysis, we monitored complex immune dynamics, identifying signatures in bone-marrow transplantation associated graft-versus-host disease. This validated and available workflow ensures comprehensive immunophenotypic analysis, data comparability and will accelerate biomarker discovery in immunomodulatory therapeutics.
biorxiv immunology 100-200-users 2018A fluorescent reporter enables instantaneous measurement of cell cycle speed in live cells, bioRxiv, 2018-12-12
AbstractPeriodicity is a fundamental property of biological oscillators such as the mitotic cell cycle. In this context, periodicity refers to the time interval between the same phases of two consecutive cell cycles. The length of this interval, or the cell cycle speed, varies widely depending on cell type and the pathophysiological conditions. The relevance of cell cycle speed in various biological contexts has not been well-studied, partially due to the lack of experimental approaches that capture this parameter. Here, we describe a genetically encoded live-cell reporter of cell cycle speed. This reporter is based on the color-changing Fluorescent Timer (FT) protein, which emits blue fluorescence when newly synthesized before maturing into a red fluorescent protein. Its ability to report cell cycle speed exploits the different half-life of the blue vs. red form of the same molecule, as predicted by mathematical modeling. When a Histone H2B-FT fusion protein is expressed at steady-state in heterogeneously dividing cells, faster-cycling cells can be distinguished from slower-cycling ones by differences in their intracellular ratio between the blue and red fluorescent wavelengths. Cell cycle perturbation experiments demonstrate that the H2B-FT is a bona fide reporter of cell cycle speed in multiple cultured cell lines. In vivo, the bluered profile faithfully tracked with known proliferation kinetics of various hematopoietic stem and progenitor cells, when expressed either from lentiviral vectors or from a targeted knock-in allele. As the H2B-FT is compatible with flow cytometry, it provides a strategy to physically separate subpopulations of live cells cycling at different rates for downstream analysis. We anticipate this system to be useful in diverse cell types and tissue contexts for dissecting the role of cell cycle speed in development and disease.
biorxiv cell-biology 100-200-users 2018Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter, bioRxiv, 2018-12-12
SummaryCell proliferation changes concomitantly with fate transitions during reprogramming, differentiation, regeneration, and oncogenesis. Methods to resolve cell cycle length heterogeneity in real-time are currently lacking. Here, we describe a genetically encoded fluorescent reporter that captures live cell cycle speed using a single measurement. This reporter is based on the color-changing Fluorescent Timer (FT) protein, which emits blue fluorescence when newly synthesized before maturing into a red fluorescent protein. We generated a mouse strain expressing an H2B-FT fusion reporter from a universally active locus, and demonstrate that faster-cycling cells can be distinguished from slower-cycling ones based on the intracellular fluorescence ratio between the FT’s blue and red states. Using this reporter, we reveal the native cell cycle speed distributions of fresh hematopoietic cells, and demonstrate its utility in analyzing cell proliferation in solid tissues. This system is broadly applicable for dissecting functional heterogeneity associated with cell cycle dynamics in complex tissues.
biorxiv cell-biology 100-200-users 2018