Biology subjects

Mansson, R.

Publications and source records attributed to Mansson, R..

11 recordsLinked to original sources

bioRxiv2025

Comparative analysis of whole genome amplification kits for single cell genome analysis

Whole genome amplification (WGA) enables detailed analysis of genomic heterogeneity at the cellular level. Here, we systematically compare two commercially available WGA technologies on single cells; Multiple Displacement Amplification in the Qiagen REPLI-g kit (MDA/REPLI-g) and Primary Template-directed Amplification in the BioSkryb ResolveDNA kit (PTA/ResolveDNA). Beyond quality control and genomic profiling, we assessed a cost-effective array-based approach as a complement to low-pass sequencing. Consistent with available benchmarks, PTA/ResolveDNA sequencing libraries consistently outperformed MDA/REPLI-g across quality metrics, including genome coverage breadth and uniformity. CNV calls from PTA-cells showed higher accuracy and sensitivity, including reliable detection of small CNVs at low sequencing depth. SNP array-based analysis corroborated sequencing-based findings, and additionally provided allelic information. Notably, CNV detection from SNP arrays matched the performance of low-pass sequencing, supporting its utility as an cost-effective approach for WGA quality control and single-cell CNV profiling.

genomics↗

bioRxiv2023

Aging is associated with functional and molecular changes in distinct hematopoietic stem cell subsets

Age is a risk factor for hematologic malignancies. Attributes of the aging hematopoietic system include increased myelopoiesis, impaired adaptive immunity, and a functional decline of the hematopoietic stem cells (HSCs) that maintain hematopoiesis. Changes in the composition of diverse HSC subsets have been suggested to be responsible for age-related alterations, however, the underlying regulatory mechanisms are incompletely understood in the context of HSC heterogeneity. In this study, we investigated how distinct HSC subsets, separated by CD49b, functionally and molecularly change their behavior with age. We demonstrate that blood lineage differentiation progressively shifts to a higher myeloid cellular output in both lymphoid-biased and myeloid-biased HSC subsets during aging. In parallel, we show that HSCs selectively undergo age-dependent gene expression and gene regulatory molecular changes in a progressive manner, which is initiated already in the pre-adult stage. Overall, our studies suggest that aging intrinsically alters both cellular and molecular properties of HSCs. HighlightsO_LIWith age a gradual shift towards myeloid differentiation occurs in both myeloid-biased and lymphoid-biased enriched HSC subsets. C_LIO_LIAge-related molecular changes preferentially occur in HSCs. C_LIO_LIFunctionally distinct HSC subsets with high transcriptional similarity can be distinguished on the epigenetic level. C_LIO_LIHSC aging is associated with a progressive increase in chromatin accessibility. C_LI

cell biology↗

bioRxiv2022

T-RHEX-RNAseq - A tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries directly from very low numbers of lysed cells.

BackgroundRNA sequencing has become the mainstay for studies of gene expression. Still, analysis of rare cells with random hexamer priming - to allow analysis of a broader range of transcripts - remains challenging. ResultsWe here describe a tagmentation-based, rRNA blocked, random hexamer primed RNAseq approach (T-RHEX-RNAseq) for generating stranded RNAseq libraries from very low numbers of FACS sorted cells without RNA purification steps. ConclusionT-RHEX-RNAseq provides an easy-to-use, time efficient and automation compatible method for generating stranded RNAseq libraries from rare cells.

molecular biology↗

bioRxiv2022

FOXO dictates initiation of B cell development and myeloid restriction in common lymphoid progenitors

The development of B cells relies on an intricate network of transcription factors critical for developmental progression and lineage commitment. In the B cell developmental trajectory, a temporal switch from predominant Foxo3 to Foxo1 expression occurs at the CLP stage. Utilizing VAV-iCre mediated conditional deletion, we found that the loss of FOXO3 impaired B cell development from LMPP down to B cell precursors, while the loss of FOXO1 impaired B cell commitment and resulted in a complete developmental block at the CD25 negative proB cell stage. Strikingly, the combined loss of FOXO1 and FOXO3 resulted in the failure to restrict the myeloid potential of CLPs and the complete loss of the B cell lineage. This is underpinned by the failure to enforce the early B-lineage gene regulatory circuitry upon a predominantly pre-established open chromatin landscape. Altogether, this demonstrates that FOXO3 and FOXO1 cooperatively govern early lineage restriction and initiation of B-lineage commitment in CLPs. SUMMARYCommon lymphoid progenitors co-express the transcription factors FOXO1 and FOXO3. Removing FOXO1 and FOXO3 at this developmental stage results in regained myeloid potential, failed establishment of the early B cell gene regulatory program, and the complete loss of the B cell lineage.

immunology↗

bioRxiv2021

Linked-read whole-genome sequencing resolves common and private structural variants in multiple myeloma

Multiple myeloma (MM) is an incurable and aggressive plasma cell malignancy characterized by a complex karyotype with multiple structural variants (SVs) and copy number variations (CNVs). Linked-read whole-genome sequencing (lrWGS) allows for refined detection and reconstruction of SVs by providing long-range genetic information from standard short-read sequencing. This makes lrWGS an attractive solution for capturing the full genomic complexity of MM. Here we show that high-quality lrWGS data can be generated from low numbers of FACS sorted cells without DNA purification. Using this protocol, we analyzed FACS sorted MM cells from 37 MM patients with lrWGS. We found high concordance between lrWGS and FISH for the detection of recurrent translocations and CNVs. Outside of the regions investigated by FISH, we identified >150 additional SVs and CNVs across the cohort. Analysis of the lrWGS data allowed for resolving the structure of diverse SVs affecting the MYC and t(11;14) loci causing the duplication of genes and gene regulatory elements. In addition, we identified private SVs causing the dysregulation of genes recurrently involved in translocations with the IGH locus and show that these can alter the molecular classification of the MM. Overall, we conclude that lrWGS allows for the detection of aberrations critical for MM prognostics and provides a feasible route for providing comprehensive genetics. Implementing lrWGS could provide more accurate clinical prognostics, facilitate genomic medicine initiatives, and greatly improve the stratification of patients included in clinical trials. KEY POINTS- Linked-read WGS can be performed without DNA purification and allows for resolving the diverse structural variants found in multiple myeloma. - Linked-read WGS can, as a stand-alone assay, provide comprehensive genetics in myeloma and other diseases with complex genomes.

genetics↗

bioRxiv2021

Cell-intrinsic functions of the transcription factor Bhlhe40 in activated B cells and T follicular helper cells restrain the germinal center reaction and prevent lymphomagenesis

The generation of high-affinity antibodies against pathogens and vaccines requires the germinal center (GC) reaction - a process that relies on a complex interplay between specialized effector subsets of B and CD4 T lymphocytes - GC B cells and T follicular helper (TFH) cells. Intriguingly, several key positive regulators of the GC reaction are common for both cell types. Here, we report that the transcription factor Bhlhe40 is a crucial cell-intrinsic negative regulator affecting both the B and T cell sides of the GC reaction. In activated CD4 T cells, Bhlhe40 was required to restrain proliferation thus limiting the number of TFH cells. In B cells, Bhlhe40 executed its function in the first days after immunization by selectively restricting the generation of the earliest GC B cells but not of early memory B cells or plasmablasts. Conditional Bhlhe40 inactivation confirmed cell-autonomous functions of Bhlhe40 in both GC B and TFH cells, while the GC phenotype was further enhanced upon loss of Bhlhe40 in both cell types. This negative regulation of the GC reaction by Bhlhe40 was of crucial importance, as Bhlhe40-deficient mice with progressing age succumbed to a B cell lymphoma characterized by accumulation of monoclonal GC B-like cells and polyclonal TFH cells in various tissues.

immunology↗

bioRxiv2021

FOXO1 and FOXO3 cooperatively regulate innate lymphoid cell development

The natural killer (NK) and non-cytotoxic innate lymphoid cells (ILC) lineages play vital role in the regulation of the immune system. Yet understanding of mechanisms controlling NK/ILC development remains incomplete. The evolutionary conserved FOXO family of forkhead transcription factors are critical regulators of cellular processes. We found that the loss of FOXO1 and FOXO3 together caused impaired activation of the NK gene expression program and reduced ETS binding already at the common lymphoid progenitor (CLP) level and a block at the ILC progenitor (ILCP) to NK progenitor transition. FOXO controlled NK cell maturation in organ specific manner and their ability to respond to IL-15. At the ILCP level, disruption of the ILC lineage specific gene programs was associated with broad perturbation of the generation of the non-cytotoxic ILC subsets. We concluded that FOXO1 and FOXO3 cooperatively regulate ILC lineage specification at the progenitor level as well as the generation of mature ILCs.

cell biology↗

bioRxiv2019

Differential genomic and transcriptomic events associated with high-grade transformation of Chronic Lymphocytic Leukemia

The transformation of chronic lymphocytic leukemia (CLL) to high-grade diffuse large B-cell lymphoma (DLBCL), also called Richters Syndrome (RS), is a rare cancer with dismal prognosis. Drug discovery for RS is hampered by the lack of suitable experimental models, and effective therapies remain elusive rendering RS an area of high unmet clinical need. We performed whole genome sequencing (WGS) to interrogate paired CLL and RS samples from 17 patients enrolled in a prospective multicenter Phase 2 clinical trial (CHOP-OR) and we found that subclones affected by mutations in MAPK and PI3K pathways show a high expansion probability during transformation. We also demonstrate for the first time that non-coding mutation clusters in a PAX5 enhancer, situated 330kb upstream from the transcription initiation site, correlate with transformation. Finally, we confirm our findings by employing targeted DNA sequencing (TGS) and RNA expression profiling on an extended cohort of 38 patients.\n\nStatement of significanceThrough integrated analysis of WGS, TGS and RNA expression data, we identified drivers of transformation not previously implicated in RS, which can be targeted therapeutically and tested in the clinic. Our results have informed the design of a new clinical platform study, which is now open to recruitment in the UK.

genomics↗

bioRxiv2018

The concerted action of E2-2 and HEB is critical for early lymphoid specification

The apparition of adaptive immunity in Gnathostomata correlates with the expansion of the E-protein family to encompass E2-2, HEB and E2A. Within the family, E2-2 and HEB are more closely evolutionarily related but their concerted action in hematopoiesis remains to be explored. Here we show that the combined disruption of E2-2 and HEB results in failure to express the early lymphoid program in CLPs and a near complete block in B-cell development. In the thymus, ETPs were reduced and T-cell development perturbed, resulting in reduced CD4 T- and increased {gamma}{delta} T-cell numbers. In contrast, HSCs, erythro-myeloid progenitors and innate immune cells were unaffected showing that E2-2 and HEB are dispensable for the ancestral hematopoietic lineages. Taken together, this E-protein dependence suggests that the appearance of the full Gnathostomata E-protein repertoire was critical to reinforce the gene regulatory circuits that drove the emergence and expansion of the lineages constituting humoral immunity.

immunology↗

bioRxiv2018

High-Throughput ChIPmentation: freely scalable, single day ChIPseq data generation from very low cell-numbers

Chromatin immunoprecipitation coupled to sequencing (ChIP-seq) is widely used to map histone modifications and transcription factor binding on a genome-wide level. Here, we present high-throughput ChIPmentation (HT-ChIPmentation) that eliminates the need for DNA purification prior to library amplification and reduces reverse-crosslinking time from hours to minutes. The resulting workflow is easily established, extremely rapid, and compatible with requirements for very low numbers of FACS sorted cells, high-throughput applications and single day data generation.

molecular biology↗

bioRxiv2016

Mice deficient of Myc super-enhancer region reveal a differential control mechanism between normal and pathological growth

The gene desert upstream of the Myc oncogene on chromosome 8q24 contains susceptibility loci for several major forms of human cancer, including cancers of breast, prostate, and colon. The region shows high conservation between human and mouse and contains multiple MYC enhancers that are activated in tumor cells. However, the role of this region in normal development has not been addressed. Here we show that a 538 kb deletion of the entire MYC upstream super-enhancer region in mice results in 50 to 80% decrease in MYC expression in multiple tissues. The mice are viable and show no overt phenotype. However, they are resistant to tumorigenesis, and most normal cells isolated from them grow slowly in culture. Consistently, deletion of the 8q24 super-enhancer region perturbs Myc targets only in cultured cells, but not in vivo. These results reveal that only cells whose Myc activity is increased by serum or oncogenic driver mutations depend on the 8q24 super-enhancer region, and indicate that targeting the activity of this element is a promising strategy of cancer chemoprevention and therapy.

cancer biology↗