Cellular zinc status alters chromatin accessibility and binding of p53 to DNA.

Zn2+ is an essential metal required by approximately 850 human transcription factors. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Using ATAC-seq to profile regions of accessible chromatin coupled with transcription factor enrichment analysis, we examined how increases and decreases in the labile zinc pool affect chromatin accessibility and transcription factor enrichment. We found 685 transcription factor motifs were differentially enriched, corresponding to 507 unique transcription factors. The pattern of perturbation and the types of transcription factors were notably different at promoters versus intergenic regions, with zinc-finger transcription factors strongly enriched in intergenic regions in elevated Zn2+ To test whether ATAC-seq and transcription factor enrichment analysis predictions correlate with changes in transcription factor binding, we used ChIP-qPCR to profile six p53 binding sites. We found that for five of the six targets, p53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc alter chromatin accessibility and transcription factor binding to DNA.

Rapid P-TEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy.

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.

A transcription factor (TF) inference method that broadly measures TF activity and identifies mechanistically distinct TF networks.

TF profiler is a method of inferring transcription factor regulatory activity, i.e. when a TF is present and actively regulating transcription, directly directly from nascent sequencing assays such as PRO-seq and GRO-seq. Transcription factors orchestrate transcription and play a critical role in cellular maintenance, identity and response to external stimuli. While ChIP assays have measured DNA localization, they fall short of identifying when and where transcription factors are actively regulating transcription. Our method, on the other hand, uses RNA polymerase activity to infer TF activity across hundreds of data sets and transcription factors. Based on these classifications we identify three distinct classes of transcription factors: ubiquitous factors that play roles in cellular homeostasis, driving basal gene programs across tissues and cell types, tissue specific factors that act almost exclusively at enhancers and are themselves regulated at transcription, and stimulus responsive TFs which are regulated post-transcriptionally but act predominantly at enhancers. TF profiler is broadly applicable, providing regulatory insights on any PRO-seq sample for any transcription factor with a known binding motif.

Macrophages derived from human induced pluripotent stem cells (iPSCs) serve as a high-fidelity cellular model for investigating HIV-1, dengue, and influenza viruses.

Macrophages are important target cells for diverse viruses and thus represent a valuable system for studying virus biology. Isolation of primary human macrophages is done by culture of dissociated tissues or from differentiated blood monocytes, but these methods are both time consuming and result in low numbers of recovered macrophages. Here, we explore whether macrophages derived from human induced pluripotent stem cells (iPSCs)-which proliferate indefinitely and potentially provide unlimited starting material-could serve as a faithful model system for studying virus biology. Human iPSC-derived monocytes were differentiated into macrophages and then infected with HIV-1, dengue virus, or influenza virus as model human viruses. We show that iPSC-derived macrophages support the replication of these viruses with kinetics and phenotypes similar to human blood monocyte-derived macrophages. These iPSC-derived macrophages were virtually indistinguishable from human blood monocyte-derived macrophages based on surface marker expression (flow cytometry), transcriptomics (RNA sequencing), and chromatin accessibility profiling. iPSC lines were additionally generated from non-human primate (chimpanzee) fibroblasts. When challenged with dengue virus, human and chimpanzee iPSC-derived macrophages show differential susceptibility to infection, thus providing a valuable resource for studying the species-tropism of viruses. We also show that blood- and iPSC-derived macrophages both restrict influenza virus at a late stage of the virus lifecycle. Collectively, our results substantiate iPSC-derived macrophages as an alternative to blood monocyte-derived macrophages for the study of virus biology. IMPORTANCE: Macrophages have complex relationships with viruses: while macrophages aid in the removal of pathogenic viruses from the body, macrophages are also manipulated by some viruses to serve as vessels for viral replication, dissemination, and long-term persistence. Here, we show that iPSC-derived macrophages are an excellent model that can be exploited in virology.

Internal and external normalization of nascent RNA sequencing run-on experiments.

In experiments with significant perturbations to transcription, nascent RNA sequencing protocols are dependent on external spike-ins for reliable normalization. Unlike in RNA-seq, these spike-ins are not standardized and, in many cases, depend on a run-on reaction that is assumed to have constant efficiency across samples. To assess the validity of this assumption, we analyze a large number of published nascent RNA spike-ins to quantify their variability across existing normalization methods. Furthermore, we develop a new biologically-informed Bayesian model to estimate the error in spike-in based normalization estimates, which we term Virtual Spike-In (VSI). We apply this method both to published external spike-ins as well as using reads at the [Formula: see text] end of long genes, building on prior work from Mahat (Mol Cell 62(1):63-78, 2016. https://doi.org/10.1016/j.molcel.2016.02.025 ) and Vihervaara (Nat Commun 8(1):255, 2017. https://doi.org/10.1038/s41467-017-00151-0 ). We find that spike-ins in existing nascent RNA experiments are typically under sequenced, with high variability between samples. Furthermore, we show that these high variability estimates can have significant downstream effects on analysis, complicating biological interpretations of results.

Deconvolution of Nascent Sequencing Data Using Transcriptional Regulatory Elements.

The problem of microdissection of heterogeneous tissue samples is of great interest for both fundamental biology and biomedical research. Until now, microdissection in the form of supervised deconvolution of mixed sequencing samples has been limited to assays measuring gene expression (RNA-seq) or chromatin accessibility (ATAC-seq). We present here the first attempt at solving the supervised deconvolution problem for run-on nascent sequencing data (GRO-seq and PRO-seq), a readout of active transcription. Then, we develop a novel filtering method suited to the mixed set of promoter and enhancer regions provided by nascent sequencing, and apply best-practice standards from the RNA-seq literature, using in-silico mixtures of cells. Using these methods, we find that enhancer RNAs are highly informative features for supervised deconvolution. In most cases, simple deconvolution methods perform better than more complex ones for solving the nascent deconvolution problem. Furthermore, undifferentiated cell types confound deconvolution of nascent sequencing data, likely as a consequence of transcriptional activity over the highly open chromatin regions of undifferentiated cell types. Our results suggest that while the problem of nascent deconvolution is generally tractable, stronger approaches integrating other sequencing protocols may be required to solve mixtures containing undifferentiated celltypes.

Cellular zinc status alters chromatin accessibility and binding of transcription factor p53 to genomic sites.

Zinc (Zn2+) is an essential metal required by approximately 2500 proteins. Nearly half of these proteins act on DNA, including > 850 human transcription factors, polymerases, DNA damage response factors, and proteins involved in chromatin architecture. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Here, we examine how changes in the labile Zn2+ pool affect chromatin accessibility and transcription factor binding to DNA. We observed both increases and decreases in accessibility in different chromatin regions via ATAC-seq upon treating MCF10A cells with elevated Zn2+ or the Zn2+-specific chelator tris(2-pyridylmethyl)amine (TPA). Transcription factor enrichment analysis was used to correlate changes in chromatin accessibility with transcription factor motifs, revealing 477 transcription factor motifs that were differentially enriched upon Zn2+ perturbation. 186 of these transcription factor motifs were enriched in Zn2+ and depleted in TPA, and the majority correspond to Zn2+ finger transcription factors. We selected TP53 as a candidate to examine how changes in motif enrichment correlate with changes in transcription factor occupancy by ChIP-qPCR. Using publicly available ChIP-seq and nascent transcription datasets, we narrowed the 50,000+ ATAC-seq peaks to 2164 TP53 targets and subsequently selected 6 high-probability TP53 binding sites for testing. ChIP-qPCR revealed that for 5 of the 6 targets, TP53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc directly alter chromatin accessibility and transcription factor binding to DNA.

An intronic LINE-1 regulates IFNAR1 expression in human immune cells.

BACKGROUND: Despite their origins as selfish parasitic sequences, some transposons in the human genome have been co-opted to serve as regulatory elements, contributing to the evolution of transcriptional networks. Most well-characterized examples of transposon-derived regulatory elements derive from endogenous retroviruses (ERVs), due to the intrinsic regulatory activity of proviral long terminal repeat regions. However, one subclass of transposable elements, the Long Interspersed Nuclear Elements (LINEs), have been largely overlooked in the search for functional regulatory transposons, and considered to be broadly epigenetically repressed. RESULTS: We examined the chromatin state of LINEs by analyzing epigenomic data from human immune cells. Many LINEs are marked by the repressive H3K9me3 modification, but a subset exhibits evidence of enhancer activity in human immune cells despite also showing evidence of epigenetic repression. We hypothesized that these competing forces of repressive and activating epigenetic marks might lead to inducible enhancer activity. We investigated a specific L1M2a element located within the first intron of Interferon Alpha/Beta Receptor 1 (IFNAR1). This element shows epigenetic signatures of B cell-specific enhancer activity, despite being repressed by the Human Silencing Hub (HUSH) complex. CRISPR deletion of the element in B lymphoblastoid cells revealed that the element acts as an enhancer that regulates both steady state and interferon-inducible expression of IFNAR1. CONCLUSIONS: Our study experimentally demonstrates that an L1M2a element was co-opted to function as an interferon-inducible enhancer of IFNAR1, creating a feedback loop wherein IFNAR1 is transcriptionally upregulated by interferon signaling. This finding suggests that other LINEs may exhibit cryptic cell type-specific or context-dependent enhancer activity. LINEs have received less attention than ERVs in the effort to understand the contribution of transposons to the regulatory landscape of cellular genomes, but these are likely important, lineage-specific players in the rapid evolution of immune system regulatory networks and deserve further study.

Atlas of nascent RNA transcripts reveals enhancer to gene linkages.

Gene transcription is controlled and modulated by regulatory regions, including enhancers and promoters. These regions are abundant in unstable, non-coding bidirectional transcription. Using nascent RNA transcription data across hundreds of human samples, we identified over 800,000 regions containing bidirectional transcription. We then identify highly correlated transcription between bidirectional and gene regions. The identified correlated pairs, a bidirectional region and a gene, are enriched for disease associated SNPs and often supported by independent 3D data. We present these resources as an SQL database which serves as a resource for future studies into gene regulation, enhancer associated RNAs, and transcription factors.

Human mRNA in saliva can correctly identify individuals harboring acute infection.

The COVID-19 pandemic demonstrated the poor ability of body temperature to reliably identify SARS-CoV-2-infected individuals, an observation that has been made before in the context of other infectious diseases. While acute infection does not always cause fever, it does reliably drive host transcriptional responses as the body responds at the site of infection. These transcriptional changes can occur both in cells that are directly harboring replicating pathogens and in cells elsewhere that receive a molecular signal that infection is occurring. Here, we identify a core set of approximately 70 human genes that are together upregulated in cultured human cells infected by a broad array of viral, bacterial, and fungal pathogens. We have named these "core response" genes. In theory, transcripts from these genes could serve as biomarkers of infection in the human body, in a way that is agnostic to the specific pathogen causing infection. As such, we perform human studies to show that these infection-induced human transcripts can be measured in the saliva of people harboring different types of infections. The number of these transcripts in saliva can correctly classify infection status (whether a person harbors an infection) 91% of the time. Furthermore, in the case of SARS-CoV-2 specifically, the number of core response transcripts in saliva correctly identifies infectious individuals even when enrollees, themselves, are asymptomatic and do not know they are infected.IMPORTANCEThere are a variety of clinical and laboratory criteria available to clinicians in controlled healthcare settings to help them identify whether an infectious disease is present. However, in situations such as a new epidemic caused by an unknown infectious agent, in health screening contexts performed within communities and outside of healthcare facilities or in battlefield or potential biowarfare situations, this gets more difficult. Pathogen-agnostic methods for rapid screening and triage of large numbers of people for infection status are needed, in particular methods that might work on an easily accessible biospecimen like saliva. Here, we identify a small, core set of approximately 70 human genes whose transcripts serve as saliva-based biomarkers of infection in the human body, in a way that is agnostic to the specific pathogen causing infection.

Transcription dosage compensation does not occur in Down syndrome.

BACKGROUND: The increase in DNA copy number in Down syndrome (DS; caused by trisomy 21) has led to the DNA dosage hypothesis, which posits that the level of gene expression is proportional to the gene's DNA copy number. Yet many reports have suggested that a proportion of chromosome 21 genes are dosage compensated back towards typical expression levels (1.0×). In contrast, other reports suggest that dosage compensation is not a common mechanism of gene regulation in trisomy 21, providing support to the DNA dosage hypothesis. RESULTS: In our work, we use both simulated and real data to dissect the elements of differential expression analysis that can lead to the appearance of dosage compensation, even when compensation is demonstrably absent. Using lymphoblastoid cell lines derived from a family with an individual with Down syndrome, we demonstrate that dosage compensation is nearly absent at both nascent transcription (GRO-seq) and steady-state RNA (RNA-seq) levels. Furthermore, we link the limited apparent dosage compensation to expected allelic variation in transcription levels. CONCLUSIONS: Transcription dosage compensation does not occur in Down syndrome. Simulated data containing no dosage compensation can appear to have dosage compensation when analyzed via standard methods. Moreover, some chromosome 21 genes that appear to be dosage compensated are consistent with allele specific expression.

Nascent transcription upon interferon-α2 stimulation on human and rhesus macaque lymphoblastoid cell lines.

OBJECTIVES: The interferon-triggered innate immune response has been observed to be under strong diversifying selection to counteract the many pathogens hosts have to defend against. In particular, rewiring of gene transcription regulation allows organisms to rapidly acquire new phenotypes by removing and adding genes into the innate immune gene network. Dissecting the molecular processes by which this rewiring takes place, either by changing the DNA regulatory elements or by changing the activity of the regulators across species, is key to better understand this evolutionary process. DATA DESCRIPTION: To better comprehend the evolutionary dynamics that have occurred in the initial transcriptional response to interferon in primates, we present Precision Run-On (PRO-seq) datasets made after 1 h of interferon-α2 stimulation on human and rhesus macaque lymphoblastoid cell lines. Further, we tested the difference between using either species' cognate interferon versus using the other orthologous interferon to account for any potential impacts in the interaction of the orthologous interferons with their cellular membrane receptors. This data provides insights into the regulatory mechanisms that drive species-specific responses to environmental perturbations, such as the one driven by the interactions of pathogens and their hosts.

Mediator kinase inhibition suppresses hyperactive interferon signaling in Down syndrome.

Hyperactive interferon (IFN) signaling is a hallmark of Down syndrome (DS), a condition caused by trisomy 21 (T21); strategies that normalize IFN signaling could benefit this population. Mediator-associated kinases CDK8 and CDK19 drive inflammatory responses through incompletely understood mechanisms. Using sibling-matched cell lines with/without T21, we investigated Mediator kinase function in the context of hyperactive IFN in DS. Activation of IFN-response genes was suppressed in cells treated with the CDK8/CDK19 inhibitor cortistatin A, and this occurred through suppression of IFN-responsive transcription factor activity. Moreover, we discovered that CDK8/CDK19 affect splicing, a novel means by which Mediator kinases control gene expression. Kinase inhibition altered splicing in pathway-specific ways and selectively affected IFN-responsive gene splicing in T21 cells. To further probe Mediator kinase function, we completed cytokine screens and untargeted metabolomics experiments. Cytokines are master regulators of inflammatory responses; by screening 105 different cytokine proteins, we show that Mediator kinases help drive IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Metabolomics revealed that Mediator kinase inhibition altered core metabolic pathways, including broad up-regulation of anti-inflammatory lipid mediators. Elevated levels of lipid mediators persisted at least 24hr after Mediator kinase inhibition, and many identified lipids serve as ligands for nuclear receptors (e.g. PPAR, LXR) or G-protein coupled receptors (GPCRs; e.g. FFAR4). Notably, ligand-dependent activation of these GPCRs or nuclear receptors will propagate anti-inflammatory signaling pathways and gene expression programs, and this mechanistic link suggests that metabolic changes caused by CDK8/CDK19 inhibition can durably and independently suppress pro-inflammatory IFN responses. Collectively, our results establish that Mediator kinase inhibition antagonizes IFN signaling through transcriptional, metabolic, and cytokine responses, with implications for DS and other chronic inflammatory conditions.

Single cell analysis of transcriptome and open chromatin reveals the dynamics of hair follicle stem cell aging.

Aging is defined as the functional decline of tissues and organisms, leading to many human conditions, such as cancer, neurodegenerative diseases, and hair loss. Although stem cell exhaustion is widely recognized as a hallmark of aging, our understanding of cell state changes-specifically, the dynamics of the transcriptome and open chromatin landscape, and their relationship with aging-remains incomplete. Here we present a longitudinal, single-cell atlas of the transcriptome and open chromatin landscape for epithelia cells of the skin across various hair cycle stages and ages in mice. Our findings reveal fluctuating hair follicle stem cell (HF-SC) states, some of which are associated with the progression of the hair cycle during aging. Conversely, inner bulge niche cells display a more linear progression, seemingly less affected by the hair cycle. Further analysis of the open chromatin landscape, determined by single-cell Assay for Transposase-Accessible Chromatin (ATAC) sequencing, demonstrates that reduced open chromatin regions in HF-SCs are associated with differentiation, whereas gained open chromatin regions in HF-SCs are linked to the transcriptional control of quiescence. These findings enhance our understanding of the transcriptional dynamics in HF-SC aging and lay the molecular groundwork for investigating and potentially reversing the aging process in future experimental studies.

Transcription dosage compensation does not occur in Down syndrome.

Background: Trisomy 21, also known as Down syndrome, describes the genetic condition of having an extra copy of chromosome 21. The increase in DNA copy number has led to the "DNA dosage hypothesis", which claims that the level of gene transcription is proportional to the gene's DNA copy number. Yet many reports have suggested that a proportion of chromosome 21 genes are dosage compensated back towards typical expression levels (1.0x). In contrast, other reports suggest that dosage compensation is not a common mechanism of gene regulation in Trisomy 21, providing support to the DNA dosage hypothesis. Results: In our work, we use both simulated and real data to dissect the elements of differential expression analysis that can lead to the appearance of dosage compensation even when compensation is demonstrably absent. Using lymphoblastoid cell lines derived from a family of an individual with Down syndrome, we demonstrate that dosage compensation is nearly absent at both nascent transcription (GRO-seq) and steady-state RNA (RNA-seq) levels. Conclusions: Transcriptional dosage compensation does not occur in Down syndrome. Simulated data containing no dosage compensation can appear to have dosage compensation when analyzed via standard methods. Moreover, some chromosome 21 genes that appear to be dosage compensated are consistent with allele specific expression.

Aging disrupts gene expression timing during muscle regeneration.

Skeletal muscle function and regenerative capacity decline during aging, yet factors driving these changes are incompletely understood. Muscle regeneration requires temporally coordinated transcriptional programs to drive myogenic stem cells to activate, proliferate, fuse to form myofibers, and to mature as myonuclei, restoring muscle function after injury. We assessed global changes in myogenic transcription programs distinguishing muscle regeneration in aged mice from young mice by comparing pseudotime trajectories from single-nucleus RNA sequencing of myogenic nuclei. Aging-specific differences in coordinating myogenic transcription programs necessary for restoring muscle function occur following muscle injury, likely contributing to compromised regeneration in aged mice. Differences in pseudotime alignment of myogenic nuclei when comparing aged with young mice via dynamic time warping revealed pseudotemporal differences becoming progressively more severe as regeneration proceeds. Disruptions in timing of myogenic gene expression programs may contribute to incomplete skeletal muscle regeneration and declines in muscle function as organisms age.

Determination of steady-state transcriptome modifications associated with repeated homotypic stress in the rat rostral posterior hypothalamic region.

Chronic stress is epidemiologically correlated with physical and psychiatric disorders. Whereas many animal models of chronic stress induce symptoms of psychopathology, repeated homotypic stressors to moderate intensity stimuli typically reduce stress-related responses with fewer, if any, pathological symptoms. Recent results indicate that the rostral posterior hypothalamic (rPH) region is a significant component of the brain circuitry underlying response reductions (habituation) associated with repeated homotypic stress. To test whether posterior hypothalamic transcriptional regulation associates with the neuroendocrine modifications induced by repeated homotypic stress, RNA-seq was performed in the rPH dissected from adult male rats that experienced either no stress, 1, 3, or 7 stressful loud noise exposures. Plasma samples displayed reliable increases of corticosterone in all stressed groups, with the smallest increase in the group exposed to 7 loud noises, indicating significant habituation compared to the other stressed groups. While few or no differentially expressed genes were detected 24-h after one or three loud noise exposures, relatively large numbers of transcripts were differentially expressed between the group exposed to 7 loud noises when compared to the control or 3-stress groups, respectively, which correlated with the corticosterone response habituation observed. Gene ontology analyses indicated multiple significant functional terms related to neuron differentiation, neural membrane potential, pre- and post-synaptic elements, chemical synaptic transmission, vesicles, axon guidance and projection, glutamatergic and GABAergic neurotransmission. Some of the differentially expressed genes (Myt1l, Zmat4, Dlx6, Csrnp3) encode transcription factors that were independently predicted by transcription factor enrichment analysis to target other differentially regulated genes in this study. A similar experiment employing in situ hybridization histochemical analysis in additional animals validated the direction of change of the 5 transcripts investigated (Camk4, Gabrb2, Gad1, Grin2a and Slc32a) with a high level of temporal and regional specificity for the rPH. In aggregate, the results suggest that distinct patterns of gene regulation are obtained in response to a repeated homotypic stress regimen; they also point to a significant reorganization of the rPH region that may critically contribute to the phenotypic modifications associated with repeated homotypic stress habituation.

Rapid PTEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy.

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates PTEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

Characterizing Primary transcriptional responses to short term heat shock in paired fraternal lymphoblastoid lines with and without Down syndrome.

Heat shock stress induces genome wide changes in transcription regulation, activating a coordinated cellular response to enable survival. Using publicly available transcriptomic and proteomic data sets comparing individuals with and without trisomy 21, we noticed many heat shock genes are up-regulated in blood samples from individuals with trisomy 21. Yet no major heat shock response regulating transcription factor is encoded on chromosome 21, leaving it unclear why trisomy 21 itself would cause a heat shock response, or how it would impact the ability of blood cells to subsequently respond when faced with heat shock stress. To explore these issues in a context independent of any trisomy 21 associated co-morbidities or developmental differences, we characterized the response to heat shock of two lymphoblastoid cell lines derived from brothers with and without trisomy 21. To carefully compare the chromatin state and the transcription status of these cell lines, we measured nascent transcription, chromatin accessibility, and single cell transcript levels in the lymphoblastoid cell lines before and after acute heat shock treatment. The trisomy 21 cells displayed a more robust heat shock response after just one hour at 42°C than the matched disomic cells. We suggest multiple potential mechanisms for this increased heat shock response in lymphoblastoid cells with trisomy 21 including the possibility that cells with trisomy 21 may exist in a hyper-reactive state due to chronic stresses. Whatever the mechanism, abnormal heat shock response in individuals with Down syndrome may hobble immune responses during fever and contribute to health problems in these individuals.