Biology subjects

Weiss, M. J.

Publications and source records attributed to Weiss, M. J..

14 recordsLinked to original sources

bioRxiv2026

Endothelial Cell Secretome Alterations Induced by Inflammatory Stress

Endothelial cells detect pathogens through pattern recognition receptors, such as Toll-like receptor 4 (TLR4), which triggers the synthesis and secretion of molecules that initiate the innate immune response. Proteins bearing signal peptides are secreted through the classical endoplasmic reticulum (ER)-Golgi-dependent route, whereas select signal-peptide-lacking cytoplasmic proteins are secreted via less well-characterized ER-Golgi-independent mechanisms, collectively termed unconventional cytoplasmic protein secretion (UCPS). To systematically characterize the secretome of human umbilical vein-derived endothelial cells (HUVECs) and delineate the contribution of UCPS, we performed deep quantitative proteomics on HUVEC cell lysates and conditioned medium before and after TLR4 stimulation with lipopolysaccharide (LPS). Of 5205 proteins detected in either fraction, 381 were enriched in the conditioned medium and therefore classified as secreted. Of these, 333 proteins (87.4%) were secreted via the conventional pathway, and 48 (12.6%) were secreted via UCPS, 43 of which were not previously associated with this process. Predicted functions of UCPS-secreted proteins include redox regulation, proteostasis, cytoskeletal remodeling, and innate immune signaling. We confirmed that -globin (HBA1), which functions as a redox sensor and regulator of nitric oxide in endothelial cells, is secreted constitutively by UCPS and at higher levels following inflammatory activation. Notably, UCPS cargo identity showed poor concordance with current computational predictors, underscoring the need for empirical datasets. Overall, our findings suggest that the HUVEC secretome includes both conventionally and unconventionally secreted proteins that regulate coagulation, angiogenesis, and immune function. Our findings establish a high-quality secretome dataset for HUVECs, providing a novel resource for future efforts to define the molecular determinants governing UCPS cargo selection and trafficking related to endothelial cell function.

cell biology↗

bioRxiv2025

Divergence between transcriptomes and chromatin accessibility during differentiation from a bipotential progenitor cell population to erythroblasts and megakaryocytes

Changes in gene expression drive differentiation along distinct cell lineages, and these shifts in gene expression are associated with alterations in chromatin accessibility and modifications reflecting activation or repression. We used deep sequencing of polyA+ RNA to map the transcriptomes of the megakaryocyte-erythroid progenitor (MEP) and cells of its two daughter lineages, erythroblasts (ERY) and megakaryocytes (MEG) in mice to reveal insights into differentiation. Transcriptome comparisons revealed that MEPs already expressed much of the MEG program while continuing to express genes associated with parallel myeloid lineages. By contrast, ERY underwent an extensive program of gene induction along with repression of pan-hematopoietic and MEG genes. Maps of transcription factor (TF) occupancy also indicated distinct modes of regulation for the MEG and ERY programs, with MEG genes preferentially occupied by hematopoietic TFs in multipotent progenitors and continued occupancy post-commitment, in contrast to erythroid genes that were primarily occupied in committed ERY. Previous work had indicated a surprising discordance in the clustering of MEP with other hematopoietic cell types by RNA-seq versus chromatin states. We combined the differential expression data with chromatin accessibility across blood cell types to identify trends that contribute to this discordance. Specifically, candidate cis-regulatory elements (cCREs) in some ERY-specific genes were precociously actuated in the bipotential cell populations, and some other genes were expressed in both the MEP population and MEG but their cCREs have less chromatin accessibility in MEP. This discordance in cell type clustering by different modalities of functional genomics may reflect the different contributions of subpopulations in the MEP to the different modalities measured.

genomics↗

bioRxiv2024

Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease

Sickle cell disease (SCD) is a common severe blood disorder, caused by one major point mutation in the HBB gene. Current pharmacotherapies are only partially effective and potentially curative allogeneic hematopoietic stem cell transplantation (HSCT) is associated with immune toxicities. Genome editing of autologous patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers a potentially curative approach to treat SCD and circumvents some problems associated with allogeneic HSCT. Although the FDA has released guidelines for evaluating genome editing risks, it remains unclear how to best to assess the preclinical safety and efficacy of genome-edited cellular drug products to prepare for a clinical trial. Here we describe rigorous pre-clinical characterization and optimization of a therapeutic {gamma}-globin gene promoter editing strategy that supported an investigational new drug (IND) application cleared by the FDA. We compared targets in the {gamma}-globin promoter and BCL11A erythroid-specific enhancer, identified a lead candidate that potently induces HbF, and tested our approach in mobilized CD34+ HSPCs from normal donors and individuals with SCD. We observed efficient editing, induction of HbF to levels predicted to be therapeutic, and reduction of sickling in red blood cells derived from edited HSPCs. With single-cell western and RNA-seq analyses, we defined the heterogeneity and specificity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased genome-wide off-target discovery followed by multiplexed targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new discoveries on ex vivo genome editing of HSCs towards clinical trials for treating SCD and other blood disorders.

genomics↗

bioRxiv2023

Cross-species regulatory landscapes and elements revealed by novel joint systematic integration of human and mouse blood cell epigenomes

Knowledge of locations and activities of cis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state Regulatory Potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbored distinctive transcription factor binding motifs that were similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we showed that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.

genomics↗

bioRxiv2023

Multiplex Base Editing to Protect from CD33-Directed Therapy: Implications for Immune and Gene Therapy

On-target toxicity to normal cells is a major safety concern with targeted immune and gene therapies. Here, we developed a base editing (BE) approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate (NHP) hematopoietic stem and progenitor cells (HSPCs) protects from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo, thus demonstrating potential for novel immunotherapies with reduced off-leukemia toxicity. For broader applications to gene therapies, we demonstrated highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in NHPs. In vitro, dual gene-edited cells could be enriched via treatment with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO). Together, our results highlight the potential of adenine base editors for improved immune and gene therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/529353v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@70c99dorg.highwire.dtl.DTLVardef@c46ef4org.highwire.dtl.DTLVardef@505fd2org.highwire.dtl.DTLVardef@c30b8c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Differential UBE2H-CTLH E2-E3 ubiquitylation modules regulate erythroid maturation

The development of haematopoietic stem cells into mature erythrocytes - erythropoiesis - is a controlled process characterized by cellular reorganisation and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin (UB) system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the UB system is poorly understood. By measuring the proteomic landscape of in vitro human erythropoiesis models, we found dynamic differential expression of subunits of the CTLH E3 ubiquitin ligase complex that formed distinct maturation stage-dependent assemblies of structurally homologous RANBP9-and RANBP10-CTLH complexes. Moreover, protein abundance of CTLHs cognate E2-conjugating enzyme UBE2H increased during terminal differentiation, which depended on catalytically active CTLH E3 complexes. CRISPR-Cas9 mediated inactivation of all CTLH E3 assemblies by targeting the catalytic subunit MAEA, or UBE2H, triggered spontaneous and accelerated maturation of erythroid progenitor cells including increased heme and haemoglobin synthesis. Thus, the orderly progression of human erythropoiesis is controlled by the assembly of distinct UBE2H-CTLH modules functioning at different developmental stages.

cell biology↗

bioRxiv2021

The SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

Acute myeloid leukemia (AML) cells rely on phospho-signaling pathways to gain unlimited proliferation potential. Here, we used domain-focused CRISPR screening to identify the nuclear phosphatase SCP4 as a dependency in AML, yet this enzyme is dispensable in normal hematopoietic progenitor cells. Using CRISPR exon scanning and gene complementation assays, we show that the catalytic function of SCP4 is essential in AML. Through mass spectrometry analysis of affinity-purified complexes, we identify the kinase paralogs STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. We show that STK35 and PDIK1L function catalytically and redundantly in the same pathway as SCP4 to maintain AML proliferation and to support amino acid biosynthesis and transport. We provide evidence that SCP4 regulates STK35/PDIK1L through two distinct mechanisms: catalytic removal of inhibitory phosphorylation and by promoting kinase stability. Our findings reveal a phosphatase-kinase signaling complex that supports the pathogenesis of AML.

cancer biology↗

bioRxiv2021

Endothelial alpha globin is a nitrite reductase

Small artery vasodilation in response to hypoxia is essential for matching oxygen supply to tissue oxygen demand. One source of hypoxic dilation via nitric oxide (NO) signaling is nitrite reduction by erythrocytic hemoglobin (2{beta}2). However, the alpha subunit of hemoglobin is also expressed in resistance artery endothelium and localized to myoendothelial junctions, a subcellular domain that contacts underlying vascular smooth muscle cells. We hypothesized that nitrite reduction mediated by endothelial alpha globin may occur at myoendothelial junctions to regulate hypoxic vasodilation. To test this concept, we created two novel mouse strains: one lacking alpha globin specifically in endothelium (EC Hba1{Delta}/{Delta}) and one where alpha globin is mutated such that its inhibitory association with endothelial NO synthase (eNOS) is prevented (Hba1WT/{Delta}36-39). In EC Hba1{Delta}/{Delta} or Hba1WT/{Delta}36-39 mice hemoglobin levels, hematocrit and erythrocyte counts were unchanged from littermate controls. Loss of the full alpha globin protein from the endothelium in the EC Hba1{Delta}/{Delta} model was associated with decreased exercise capacity and decreased intracellular nitrite utilization in hypoxic conditions. These effects were not seen in Hba1WT/{Delta}36-39 animals. Hypoxia induced vasodilation was decreased by 60% in isolated thoracodorsal arteries from EC Hba1{Delta}/{Delta}, while infusion of erythrocytes only partially rescued the dilatory response. Lastly, unlike other models where blood pressure is decreased, EC Hba1{Delta}/{Delta} blood pressure was not altered in response to hypoxia. Overall, we conclude that alpha globin in the resistance artery endothelium can act as a nitrite reductase to provide a local vasodilatory response to hypoxia.

physiology↗

bioRxiv2020

ZNF410 uniquely activates the NuRD component CHD4 to silence fetal hemoglobin expression

Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA binding protein that in human erythroid cells directly and measurably activates only one gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplant settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells whose perturbation might offer new therapeutic opportunities in the treatment of hemoglobinopathies. HighlightsO_LIA CRISPR screen implicates ZNF410 in fetal globin gene repression C_LIO_LIThe CHD4 gene is the singular direct ZNF410 target in erythroid cells C_LIO_LIThe fetal globin genes are exquisitely sensitive to CHD4 levels C_LIO_LIFive C2H2 zinc fingers of ZNF410 recognize the major groove of a 14 base pair sequence C_LI

molecular biology↗

bioRxiv2020

Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing

Genome editing has promising therapeutic potential for genetic diseases and cancer (1, 2). However, the most practicable current approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of structural chromosomal abnormalities. Here, we show that a catastrophic mutational process called chromothripsis is a previously unappreciated consequence of CRISPR-Cas9-mediated DSBs. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer (3-6). Using model cell systems and a genome editing protocol similar to ones in clinical trials (7) (NCT03655678, NCT03745287) we show that CRISPR-Cas9-mediated DNA breaks generate abnormal nuclear structures--micronuclei and chromosome bridges--that trigger chromothripsis. Chromothripsis is an on-target toxicity that may be minimized by cell manipulation protocols or screening but cannot be completely avoided in many genome editing applications.

genetics↗

bioRxiv2020

Integrative proteomics reveals principles of dynamic phospho-signaling networks in human erythropoiesis

Human erythropoiesis is exquisitely controlled at multiple levels and its dysregulation leads to numerous human diseases. Despite many functional studies focused on classical regulators, we lack a global, system-wide understanding of post-translational mechanisms coordinating erythroid maturation. Using the latest advances in mass spectrometry (MS)-based proteomics we comprehensively investigate the dynamics of protein and post-translational regulation of in vitro reconstituted CD34+ HSPC-derived erythropoiesis. This quantifies and dynamically tracks 7,400 proteins and 27,000 phosphorylation sites. Our data reveals differential temporal protein expression encompassing most protein classes and numerous post-translational regulatory cascades. Drastic cell surface remodeling across erythropoiesis include numerous orchestrated changes in solute carriers, providing new stage-specific markers. The dynamic phosphoproteomes combined with a kinome-targeting CRISPR/Cas9 screen reveal coordinated networks of erythropoietic kinases and downregulation of MAPK signaling subsequent to c-Kit attenuation as key drivers of maturation. Our global view of erythropoiesis establishes a central role of post-translational regulation in terminal differentiation.

systems biology↗

bioRxiv2019

Systematic integration of GATA transcription factors and epigenomes via IDEAS paints the regulatory landscape of mouse hematopoietic cells

Members of the GATA family of transcription factors play key roles in the differentiation of specific cell lineages by regulating the expression of target genes. Three GATA factors play distinct roles in hematopoietic differentiation. In order to better understand how these GATA factors function to regulate genes throughout the genome, we are studying the epigenomic and transcriptional landscapes of hematopoietic cells in a model-driven, integrative fashion. We have formed the collaborative multi-lab VISION project to conduct ValIdated Systematic IntegratiON of epigenomic data in mouse and human hematopoiesis. The epigenomic data included nuclease accessibility in chromatin, CTCF occupancy, and histone H3 modifications for twenty cell types covering hematopoietic stem cells, multilineage progenitor cells, and mature cells across the blood cell lineages of mouse. The analysis used the Integrative and Discriminative Epigenome Annotation System (IDEAS), which learns all common combinations of features (epigenetic states) simultaneously in two dimensions - along chromosomes and across cell types. The result is a segmentation that effectively paints the regulatory landscape in readily interpretable views, revealing constitutively active or silent loci as well as the loci specifically induced or repressed in each stage and lineage. Nuclease accessible DNA segments in active chromatin states were designated candidate cis-regulatory elements in each cell type, providing one of the most comprehensive registries of candidate hematopoietic regulatory elements to date. Applications of VISION resources are illustrated for regulation of genes encoding GATA1, GATA2, GATA3, and Ikaros. VISION resources are freely available from our website http://usevision.org.

genomics↗

bioRxiv2019

An integrative view of the regulatory and transcriptional landscapes in mouse hematopoiesis

Thousands of epigenomic datasets have been generated in the past decade, but it is difficult for researchers to effectively utilize all the data relevant to their projects. Systematic integrative analysis can help meet this need, and the VISION project was established for ValIdated Systematic IntegratiON of epigenomic data in hematopoiesis. Here, we systematically integrated extensive data recording epigenetic features and transcriptomes from many sources, including individual laboratories and consortia, to produce a comprehensive view of the regulatory landscape of differentiating hematopoietic cell types in mouse. By employing IDEAS as our Integrative and Discriminative Epigenome Annotation System, we identified and assigned epigenetic states simultaneously along chromosomes and across cell types, precisely and comprehensively. Combining nuclease accessibility and epigenetic states produced a set of over 200,000 candidate cis-regulatory elements (cCREs) that efficiently capture enhancers and promoters. The transitions in epigenetic states of these cCREs across cell types provided insights into mechanisms of regulation, including decreases in numbers of active cCREs during differentiation of most lineages, transitions from poised to active or inactive states, and shifts in nuclease accessibility of CTCF-bound elements. Regression modeling of epigenetic states at cCREs and gene expression produced a versatile resource to improve selection of cCREs potentially regulating target genes. These resources are available from our VISION website (usevision.org) to aid research in genomics and hematopoiesis.

genomics↗

bioRxiv2018

Unc 51-like autophagy-activating kinase (ULK1) mediates clearance of free {alpha}-globin in {beta}-thalassemia

Erythroid maturation is coordinated to maximize the production of hemoglobin A heterotetramers (2{beta}2) and minimize the accumulation of potentially toxic free - or {beta}-globin subunits. In {beta}-thalassemia, mutations in the {beta}-globin gene cause a build-up of free -globin, which forms intracellular precipitates that impair erythroid cell maturation and viability. Protein quality-control systems mitigate {beta}-thalassemia pathophysiology by degrading toxic free -globin. We show that loss of the Unc 51-like autophagy-activating kinase gene Ulk1 in {beta}-thalassemic mice reduces autophagic clearance of -globin in red cell precursors and exacerbates disease phenotypes, whereas inactivation of the canonical autophagy gene Atg5 has minimal effects. Systemic treatment with rapamycin to inhibit the ULK1 inhibitor mTORC1 reduces -globin precipitates and lessens pathologies in {beta}-thalassemic mice, but not in those lacking Ulk1. Similarly, rapamycin reduces free -globin accumulation in erythroblasts derived from {beta}-thalassemic patient CD34+ hematopoietic progenitors. Our findings identify a new, drug-regulatable pathway for ameliorating {beta}-thalassemia.\n\nOne Sentence SummaryRapamycin alleviates {beta}-thalassemia by stimulating ULK1-dependent autophagy of toxic free -globin.

cell biology↗