The severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) disease (COVID-19) pandemic has caused millions of deaths worldwide. Genome-wide association studies identified the 3p21.31 region as ...conferring a twofold increased risk of respiratory failure. Here, using a combined multiomics and machine learning approach, we identify the gain-of-function risk A allele of an SNP, rs17713054G>A, as a probable causative variant. We show with chromosome conformation capture and gene-expression analysis that the rs17713054-affected enhancer upregulates the interacting gene, leucine zipper transcription factor like 1 (LZTFL1). Selective spatial transcriptomic analysis of lung biopsies from patients with COVID-19 shows the presence of signals associated with epithelial-mesenchymal transition (EMT), a viral response pathway that is regulated by LZTFL1. We conclude that pulmonary epithelial cells undergoing EMT, rather than immune cells, are likely responsible for the 3p21.31-associated risk. Since the 3p21.31 effect is conferred by a gain-of-function, LZTFL1 may represent a therapeutic target.
The genome is organized via CTCF-cohesin-binding sites, which partition chromosomes into 1-5 megabase (Mb) topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). ...Here we examined in vivo an ∼80 kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ∼1 Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF-cohesin sites that are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF-cohesin boundary extends the sub-TAD to adjacent upstream CTCF-cohesin-binding sites. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF-cohesin boundaries in this sub-TAD delimit the region of chromatin to which enhancers have access and within which they interact with receptive promoters.
Enhancer elements are a key regulatory feature of many important genes. Several general features including the presence of specific histone modifications are used to demarcate potentially active ...enhancers. Here we reveal that putative enhancers marked with H3 lysine 79 (H3K79) di or trimethylation (me2/3) (which we name H3K79me2/3 enhancer elements or KEEs) can be found in multiple cell types. Mixed lineage leukemia gene (MLL) rearrangements (MLL-r) such as MLL-AF4 are a major cause of incurable acute lymphoblastic leukemias (ALL). Using the DOT1L inhibitor EPZ-5676 in MLL-AF4 leukemia cells, we show that H3K79me2/3 is required for maintaining chromatin accessibility, histone acetylation and transcription factor binding specifically at KEEs but not non-KEE enhancers. We go on to show that H3K79me2/3 is essential for maintaining enhancer-promoter interactions at a subset of KEEs. Together, these data implicate H3K79me2/3 as having a functional role at a subset of active enhancers in MLL-AF4 leukemia cells.
In higher eukaryotes, many genes are regulated by enhancers that are 10
-10
base pairs (bp) away from the promoter. Enhancers contain transcription-factor-binding sites (which are typically around ...7-22 bp), and physical contact between the promoters and enhancers is thought to be required to modulate gene expression. Although chromatin architecture has been mapped extensively at resolutions of 1 kilobase and above; it has not been possible to define physical contacts at the scale of the proteins that determine gene expression. Here we define these interactions in detail using a chromosome conformation capture method (Micro-Capture-C) that enables the physical contacts between different classes of regulatory elements to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show that transcription factors have an important role in the maintenance of the contacts between enhancers and promoters. Our data show that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. This supports a model in which chromatin loop extrusion
is dependent on cohesin loading at active promoters and enhancers, which explains the formation of tissue-specific chromatin domains without changes in CTCF binding.
Many single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new ...regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements.
The promoters of mammalian genes are commonly regulated by multiple distal enhancers, which physically interact within discrete chromatin domains. How such domains form and how the regulatory ...elements within them interact in single cells is not understood. To address this we developed Tri-C, a new chromosome conformation capture (3C) approach, to characterize concurrent chromatin interactions at individual alleles. Analysis by Tri-C identifies heterogeneous patterns of single-allele interactions between CTCF boundary elements, indicating that the formation of chromatin domains likely results from a dynamic process. Within these domains, we observe specific higher-order structures that involve simultaneous interactions between multiple enhancers and promoters. Such regulatory hubs provide a structural basis for understanding how multiple cis-regulatory elements act together to establish robust regulation of gene expression.
The successful development and ongoing functioning of complex organisms depend on the faithful execution of the genetic code. A critical step in this process is the correct spatial and temporal ...expression of genes. The highly orchestrated transcription of genes is controlled primarily by
cis-
regulatory elements: promoters, enhancers, and insulators. The medical importance of this key biological process can be seen by the frequency with which mutations and inherited variants that alter
cis-
regulatory elements lead to monogenic and complex diseases and cancer. Here, we provide an overview of the methods available to characterize and perturb gene regulatory circuits. We then highlight mechanisms through which regulatory rewiring contributes to disease, and conclude with a perspective on how our understanding of gene regulation can be used to improve human health.
The chromatin remodeller ATRX interacts with the histone chaperone DAXX to deposit the histone variant H3.3 at sites of nucleosome turnover. ATRX is known to bind repetitive, heterochromatic regions ...of the genome including telomeres, ribosomal DNA and pericentric repeats, many of which are putative G-quadruplex forming sequences (PQS). At these sites ATRX plays an ancillary role in a wide range of nuclear processes facilitating replication, chromatin modification and transcription. Here, using an improved protocol for chromatin immunoprecipitation, we show that ATRX also binds active regulatory elements in euchromatin. Mutations in ATRX lead to perturbation of gene expression associated with a reduction in chromatin accessibility, histone modification, transcription factor binding and deposition of H3.3 at the sequences to which it normally binds. In erythroid cells where downregulation of α-globin expression is a hallmark of ATR-X syndrome, perturbation of chromatin accessibility and gene expression occurs in only a subset of cells. The stochastic nature of this process suggests that ATRX acts as a general facilitator of cell specific transcriptional and epigenetic programmes, both in heterochromatin and euchromatin.
Dynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). However, despite years of studies, our knowledge about the protein copy ...number of TFs in the nucleus is limited. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first gene regulatory network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs’ cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that, in the nucleus, co-repressors are dramatically more abundant than co-activators at the protein level, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.
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•Absolute quantification of endogenous transcription factors in human erythropoiesis•Co-repressors are in large excess while co-activators are limiting in the nucleus•Quantitative model of erythropoiesis integrating changes in protein and mRNA amounts•A website for human erythropoiesis transcription factors proteins and transcripts
Protein stoichiometry in the nucleus is mostly unknown. Gillespie et al. use targeted mass spectrometry to quantify over a hundred transcription factors during human erythropoiesis. The data reveal co-repressors are on average 100 times more abundant than co-activators. Furthermore, a quantitative model of erythropoiesis integrating mRNA and protein abundances is generated.
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