Comprehensive In Vivo Interrogation Reveals Phenotypic Impact of Human Enhancer Variants.

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.

Risk SNP-Mediated Promoter-Enhancer Switching Drives Prostate Cancer through lncRNA PCAT19.

The prostate cancer (PCa) risk-associated SNP rs11672691 is positively associated with aggressive disease at diagnosis. We showed that rs11672691 maps to the promoter of a short isoform of long noncoding RNA PCAT19 (PCAT19-short), which is in the third intron of the long isoform (PCAT19-long). The risk variant is associated with decreased and increased levels of PCAT19-short and PCAT19-long, respectively. Mechanistically, the risk SNP region is bifunctional with both promoter and enhancer activity. The risk variants of rs11672691 and its LD SNP rs887391 decrease binding of transcription factors NKX3.1 and YY1 to the promoter of PCAT19-short, resulting in weaker promoter but stronger enhancer activity that subsequently activates PCAT19-long. PCAT19-long interacts with HNRNPAB to activate a subset of cell-cycle genes associated with PCa progression, thereby promoting PCa tumor growth and metastasis. Taken together, these findings reveal a risk SNP-mediated promoter-enhancer switching mechanism underlying both initiation and progression of aggressive PCa.

Progressive Loss of Function in a Limb Enhancer during Snake Evolution.

The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution. PAPERCLIP.

Deciphering the multi-scale, quantitative cis-regulatory code.

Uncovering the cis-regulatory code that governs when and how much each gene is transcribed in a given genome and cellular state remains a central goal of biology. Here, we discuss major layers of regulation that influence how transcriptional outputs are encoded by DNA sequence and cellular context. We first discuss how transcription factors bind specific DNA sequences in a dosage-dependent and cooperative manner and then proceed to the cofactors that facilitate transcription factor function and mediate the activity of modular cis-regulatory elements such as enhancers, silencers, and promoters. We then consider the complex and poorly understood interplay of these diverse elements within regulatory landscapes and its relationships with chromatin states and nuclear organization. We propose that a mechanistically informed, quantitative model of transcriptional regulation that integrates these multiple regulatory layers will be the key to ultimately cracking the cis-regulatory code.

Systematic analysis of intrinsic enhancer-promoter compatibility in the mouse genome.

Gene expression is in part controlled by cis-regulatory elements (CREs) such as enhancers and repressive elements. Anecdotal evidence has indicated that a CRE and a promoter need to be biochemically compatible for promoter regulation to occur, but this compatibility has remained poorly characterized in mammalian cells. We used high-throughput combinatorial reporter assays to test thousands of CRE-promoter pairs from three Mb-sized genomic regions in mouse cells. This revealed that CREs vary substantially in their promoter compatibility, ranging from striking specificity to broad promiscuity. More than half of the tested CREs exhibit significant promoter selectivity. Housekeeping promoters tend to have similar CRE preferences, but other promoters exhibit a wide diversity of compatibilities. Higher-order transcription factors (TF) motif combinations may account for compatibility. CRE-promoter selectivity does not correlate with looping interactions in the native genomic context, suggesting that chromatin folding and compatibility are two orthogonal mechanisms that confer specificity to gene regulation.

High Amount of Transcription Factor IRF8 Engages AP1-IRF Composite Elements in Enhancers to Direct Type 1 Conventional Dendritic Cell Identity.

Development and function of conventional dendritic cell (cDC) subsets, cDC1 and cDC2, depend on transcription factors (TFs) IRF8 and IRF4, respectively. Since IRF8 and IRF4 can each interact with TF BATF3 at AP1-IRF composite elements (AICEs) and with TF PU.1 at Ets-IRF composite elements (EICEs), it is unclear how these factors exert divergent actions. Here, we determined the basis for distinct effects of IRF8 and IRF4 in cDC development. Genes expressed commonly by cDC1 and cDC2 used EICE-dependent enhancers that were redundantly activated by low amounts of either IRF4 or IRF8. By contrast, cDC1-specific genes relied on AICE-dependent enhancers, which required high IRF concentrations, but were activated by either IRF4 or IRF8. IRF8 was specifically required only by a minority of cDC1-specific genes, such as Xcr1, which could distinguish between IRF8 and IRF4 DNA-binding domains. Thus, these results explain how BATF3-dependent Irf8 autoactivation underlies emergence of the cDC1-specific transcriptional program.

Viral cis-regulatory elements as sensors of cellular states and environmental cues.

To withstand a hostile cellular environment and replicate, viruses must sense, interpret, and respond to many internal and external cues. Retroviruses and DNA viruses can intercept these cues impinging on host transcription factors via cis-regulatory elements (CREs) in viral genomes, allowing them to sense and coordinate context-specific responses to varied signals. Here, we explore the characteristics of viral CREs, the classes of signals and host transcription factors that regulate them, and how this informs outcomes of viral replication, immune evasion, and latency. We propose that viral CREs constitute central hubs for signal integration from multiple pathways and that sequence variation between viral isolates can rapidly rewire sensing mechanisms, contributing to the variability observed in patient outcomes.

SpliceVarDB: A comprehensive database of experimentally validated human splicing variants.

Variants that alter gene splicing are estimated to comprise up to a third of all disease-causing variants, yet they are hard to predict from DNA sequencing data alone. To overcome this, many groups are incorporating RNA-based analyses, which are resource intensive, particularly for diagnostic laboratories. There are thousands of functionally validated variants that induce mis-splicing; however, this information is not consolidated, and they are under-represented in ClinVar, which presents a barrier to variant interpretation and can result in duplication of validation efforts. To address this issue, we developed SpliceVarDB, an online database consolidating over 50,000 variants assayed for their effects on splicing in over 8,000 human genes. We evaluated over 500 published data sources and established a spliceogenicity scale to standardize, harmonize, and consolidate variant validation data generated by a range of experimental protocols. According to the strength of their supporting evidence, variants were classified as "splice-altering" (∼25%), "not splice-altering" (∼25%), and "low-frequency splice-altering" (∼50%), which correspond to weak or indeterminate evidence of spliceogenicity. Importantly, 55% of the splice-altering variants in SpliceVarDB are outside the canonical splice sites (5.6% are deep intronic). These variants can support the variant curation diagnostic pathway and can be used to provide the high-quality data necessary to develop more accurate in silico splicing predictors. The variants are accessible through an online platform, SpliceVarDB, with additional features for visualization, variant information, in silico predictions, and validation metrics. SpliceVarDB is a very large collection of splice-altering variants and is available at https://splicevardb.org.

Enhancer Features that Drive Formation of Transcriptional Condensates.

Enhancers are DNA elements that are bound by transcription factors (TFs), which recruit coactivators and the transcriptional machinery to genes. Phase-separated condensates of TFs and coactivators have been implicated in assembling the transcription machinery at particular enhancers, yet the role of DNA sequence in this process has not been explored. We show that DNA sequences encoding TF binding site number, density, and affinity above sharply defined thresholds drive condensation of TFs and coactivators. A combination of specific structured (TF-DNA) and weak multivalent (TF-coactivator) interactions allows for condensates to form at particular genomic loci determined by the DNA sequence and the complement of expressed TFs. DNA features found to drive condensation promote enhancer activity and transcription in cells. Our study provides a framework to understand how the genome can scaffold transcriptional condensates at specific loci and how the universal phenomenon of phase separation might regulate this process.

Deciphering the Role of Rapidly Evolving Conserved Elements in Primate Brain Development and Exploring Their Potential Involvement in Alzheimer's Disease.

Although previous studies have identified human-specific accelerated regions as playing a key role in the recent evolution of the human brain, the characteristics and cellular functions of rapidly evolving conserved elements (RECEs) in ancestral primate lineages remain largely unexplored. Here, based on large-scale primate genome assemblies, we identify 888 RECEs that have been highly conserved in primates that exhibit significantly accelerated substitution rates in the ancestor of the Simiiformes. This primate lineage exhibits remarkable morphological innovations, including an expanded brain mass. Integrative multiomic analyses reveal that RECEs harbor sequences with potential cis-regulatory functions that are activated in the adult human brain. Importantly, genes linked to RECEs exhibit pronounced expression trajectories in the adult brain relative to the fetal stage. Furthermore, we observed an increase in the chromatin accessibility of RECEs in oligodendrocytes from individuals with Alzheimer's disease (AD) compared to that of a control group, indicating that these RECEs may contribute to brain aging and AD. Our findings serve to expand our knowledge of the genetic underpinnings of brain function during primate evolution.

Interplay between regulatory elements and chromatin topology in cellular lineage determination.

Cellular lineage determination is controlled by combinations of lineage-selective transcription factors (TFs) and associated coregulators that bind to cis-regulatory elements in DNA and regulate gene expression. The ability of these factors to regulate transcription is determined not only by their cooperativity, but also by biochemical and structural properties of the chromatin, sculpting higher-order genome organization. Here, we review recent advances in the understanding of the interplay between chromatin topology and transcription. Studies from many different fields, including adipocyte lineage determination, indicate that lineage determination and differentiation are dependent on elaborate crosstalk between cis-regulatory elements, leading to the formation of transcriptional hubs. Chromatin topology appears to provide a dynamic and supportive, rather than a deterministic, scaffold for this crosstalk.

SilenceREIN: seeking silencers on anchors of chromatin loops by deep graph neural networks.

Silencers are repressive cis-regulatory elements that play crucial roles in transcriptional regulation. Experimental methods for identifying silencers are always costly and time-consuming. Computational methods, which relies on genomic sequence features, have been introduced as alternative approaches. However, silencers do not have significant epigenomic signature. Therefore, we explore a new way to computationally identify silencers, by incorporating chromatin structural information. We propose the SilenceREIN method, which focuses on finding silencers on anchors of chromatin loops. By using graph neural networks, we extracted chromatin structural information from a regulatory element interaction network. SilenceREIN integrated the chromatin structural information with linear genomic signatures to find silencers. The predictive performance of SilenceREIN is comparable or better than other states-of-the-art methods. We performed a genome-wide scanning to systematically find silencers in human genome. Results suggest that silencers are widespread on anchors of chromatin loops. In addition, enrichment analysis of transcription factor binding motif support our prediction results. As far as we can tell, this is the first attempt to incorporate chromatin structural information in finding silencers. All datasets and source codes of SilenceREIN have been deposited in a GitHub repository (https://github.com/JianHPan/SilenceREIN).

AnchorWave: Sensitive alignment of genomes with high sequence diversity, extensive structural polymorphism, and whole-genome duplication.

Millions of species are currently being sequenced, and their genomes are being compared. Many of them have more complex genomes than model systems and raise novel challenges for genome alignment. Widely used local alignment strategies often produce limited or incongruous results when applied to genomes with dispersed repeats, long indels, and highly diverse sequences. Moreover, alignment using many-to-many or reciprocal best hit approaches conflicts with well-studied patterns between species with different rounds of whole-genome duplication. Here, we introduce Anchored Wavefront alignment (AnchorWave), which performs whole-genome duplication-informed collinear anchor identification between genomes and performs base pair-resolved global alignment for collinear blocks using a two-piece affine gap cost strategy. This strategy enables AnchorWave to precisely identify multikilobase indels generated by transposable element (TE) presence/absence variants (PAVs). When aligning two maize genomes, AnchorWave successfully recalled 87% of previously reported TE PAVs. By contrast, other genome alignment tools showed low power for TE PAV recall. AnchorWave precisely aligns up to three times more of the genome as position matches or indels than the closest competitive approach when comparing diverse genomes. Moreover, AnchorWave recalls transcription factor-binding sites at a rate of 1.05- to 74.85-fold higher than other tools with significantly lower false-positive alignments. AnchorWave complements available genome alignment tools by showing obvious improvement when applied to genomes with dispersed repeats, active TEs, high sequence diversity, and whole-genome duplication variation.

Systematic single-cell analysis reveals dynamic control of transposable element activity orchestrating the endothelial-to-hematopoietic transition.

BACKGROUND: The endothelial-to-hematopoietic transition (EHT) process during definitive hematopoiesis is highly conserved in vertebrates. Stage-specific expression of transposable elements (TEs) has been detected during zebrafish EHT and may promote hematopoietic stem cell (HSC) formation by activating inflammatory signaling. However, little is known about how TEs contribute to the EHT process in human and mouse. RESULTS: We reconstructed the single-cell EHT trajectories of human and mouse and resolved the dynamic expression patterns of TEs during EHT. Most TEs presented a transient co-upregulation pattern along the conserved EHT trajectories, coinciding with the temporal relaxation of epigenetic silencing systems. TE products can be sensed by multiple pattern recognition receptors, triggering inflammatory signaling to facilitate HSC emergence. Interestingly, we observed that hypoxia-related signals were enriched in cells with higher TE expression. Furthermore, we constructed the hematopoietic cis-regulatory network of accessible TEs and identified potential TE-derived enhancers that may boost the expression of specific EHT marker genes. CONCLUSIONS: Our study provides a systematic vision of how TEs are dynamically controlled to promote the hematopoietic fate decisions through transcriptional and cis-regulatory networks, and pre-train the immunity of nascent HSCs.

Dimethyloxalylglycine Suppresses SREBP1c and Lipogenic Gene Expressions in Hepatocytes Independently of HIF1A.

Dimethyloxalylglycine (DMOG) is a representative inhibitor of the prolyl hydroxylase domain (PHD), which mediates the degradation of hypoxia-inducible factor-1-alpha (HIF1A). DMOG exerts its pharmacological effects via the canonical pathway that involves PHD inhibition; however, it remains unclear whether DMOG affects lipogenic gene expression in hepatocytes. We aimed to elucidate the effects of DMOG on sterol regulatory element-binding protein-1c (SREBP1c), a master regulator of fatty acid synthesis in hepatocytes. DMOG treatment inhibited SREBP1c mRNA and protein expression in HepG2 and AML12 hepatocytes and reduced the transcript levels of SREBP1c-regulated lipogenic genes. A luciferase reporter assay revealed that DMOG inhibited the transcriptional activity of SREBP1c. Moreover, DMOG suppressed SREBP1c expression in mice liver. Mechanistically, treatment with DMOG enhanced the expression of HIF1A and insulin-induced gene 2 (INSIG2), which inhibits the activation of SREBP1c. However, HIF1A or INSIG2 knockdown failed to reverse the inhibitory effect of DMOG on SREBP1c expression, suggesting a redundant role of HIF1A and INSIG2 in terms of repressing SREBP1c. DMOG did not function through the canonical pathway involving inhibition of SREBP1c by PHD, highlighting the presence of non-canonical pathways that mediate its anti-lipogenic effect.

Cholesterol dynamics in rabbit liver: High-fat diet, olive oil, and synergistic dietary effects.

BACKGROUND & AIMS: Lipid metabolism disorders contribute to a range of human diseases, including liver-related pathologies. Rabbits, highly sensitive to dietary cholesterol, provide a model for understanding the development of liver disorders. Sterol regulatory element-binding protein isoform 2 (SREBP2) crucially regulates intracellular cholesterol pathways. Extra-virgin olive oil (EVOO) has shown reducing cholesterol levels and restoring liver parameters affected by HFD. The aim was to investigate the molecular impact of an HFD and supplemented with EVOO on rabbit liver cholesterol metabolism. APPROACH & RESULTS: Male rabbits were assigned to dietary cohorts, including control, acute/chronic HFD, sequential HFD with EVOO, and EVOO. Parameters such as serum lipid profiles, hepatic enzymes, body weight, and molecular analyses. After 6 months of HFD, plasma and hepatic cholesterol increased with decreased SREBP2 and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) expression. Prolonged HFD increased cholesterol levels, upregulating SREBP2 mRNA and HMGCR protein. Combining this with EVOO lowered cholesterol, increased SREBP2 mRNA, and upregulated low-density lipoprotein receptor (LDLR) expression. HFD-induced metabolic dysfunction-associated fatty liver disease was mitigated by EVOO. In conclusion, the SREBP2 system responds to dietary changes. CONCLUSIONS: In rabbits, the SREBP2 system responds to dietary changes. Acute HFD hinders cholesterol synthesis, while prolonged HFD disrupts regulation, causing SREBP2 upregulation. EVOO intake prompts LDLR upregulation, potentially enhancing cholesterol clearance and restoring hepatic alterations.

Preclinical Milestones in MECP2 Gene Transfer for Treating Rett Syndrome.

BACKGROUND: Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2). After gene transfer in mice, exogenous MeCP2 expression must be regulated to avoid dose-dependent toxicity. SUMMARY: The preclinical gene therapy literature for treating RTT illustrates a duly diligent progression that begins with proof-of-concept studies and advances toward the development of safer, regulated MECP2 viral genome designs. This design progression was partly achieved through international collaborative studies. In 2023, clinicians administered investigational gene therapies for RTT to patients a decade after the first preclinical gene therapy publications for RTT (clinical trial numbers NCT05606614 and NCT05898620). As clinicians take on a more prominent role in MECP2 gene therapy research, preclinical researchers may continue to test more nuanced hypotheses regarding the safety, efficacy, and mechanism of MECP2 gene transfer. KEY MESSAGE: This review summarizes the history of preclinical MECP2 gene transfer for treating RTT and acknowledges major contributions among colleagues in the field. The first clinical injections are a shared milestone.

Large-scale analysis of the N-terminal regulatory elements of the kinase domain in plant Receptor-like kinase family.

BACKGROUND: The N-terminal regulatory element (NRE) of Receptor-like kinases (RLKs), consisting of the juxtamembrane segment in receptor kinases (RKs) and the N-terminal extension segment in RLCKs, is a crucial component that regulates the activities of these proteins. However, the features and functions of the NRE have remained largely unexplored. Herein, we comprehensively analyze 510,233 NRE sequences in RLKs from 528 plant species, using information theory and data mining techniques to unravel their common characteristics and diversity. We also use recombinant RKs to investigate the function of the NRE in vitro. RESULTS: Our findings indicate that the majority of NRE segments are around 40-80 amino acids in length and feature a serine-rich region and a 14-amino-acid consensus sequence, 'FSYEELEKAT[D/N]NF[S/D]', which contains a characteristic α-helix and ST motif that connects to the core kinase domain. This conserved signature sequence is capable of suppressing FERONIA's kinase activity. A motif discovery algorithm identifies 29 motifs with highly conserved phosphorylation sites in RK and RLCK classes, especially the motif 'VGPWKpTGLpSGQLQKAFVTGVP' in LRR-VI-2 class. Phosphorylation of an NRE motif in an LRR-VI-2 member, MDIS1, modulates the auto-phosphorylation of its co-receptor, MIK1, indicating the potential role of NRE as a 'kinase switch' in RLK activation. Furthermore, the characterization of phosphorylatable NRE motifs improves the accuracy of predicting phosphorylatable sites. CONCLUSIONS: Our study provides a comprehensive dataset to investigate NRE segments from individual RLKs and enhances our understanding of the underlying mechanisms of RLK signal transduction and kinase activation processes in plant adaptation.

Sterol Regulatory Element Binding Protein 1: A Mediator for High-Fat Diet-Induced Hepatic Gluconeogenesis and Glucose Intolerance in Fish.

BACKGROUND: Sterol regulatory element binding protein (SREBP) 1 is considered to be a crucial regulator for lipid synthesis in vertebrates. However, whether SREBP1 could regulate hepatic gluconeogenesis under high-fat diet (HFD) condition is still unknown, and the underlying mechanism is also unclear. OBJECTIVES: This study aimed to determine gluconeogenesis-related gene and protein expressions in response to HFD in large yellow croaker and explore the role and mechanism of SREBP1 in regulating the related transcription and signaling. METHODS: Croakers (mean weight, 15.61 ± 0.10 g) were fed with diets containing 12% crude lipid [control diet (ND)] or 18% crude lipid (HFD) for 10 weeks. The glucose tolerance, insulin tolerance, hepatic gluconeogenesis-related genes, and proteins expressions were determined. To explore the role of SREBP1 in HFD-induced gluconeogenesis, SREBP1 was inhibited by pharmacologic inhibitor (fatostatin) or genetic knockdown in croaker hepatocytes under palmitic acid (PA) condition. To explore the underlying mechanism, luciferase reporter and chromatin immunoprecipitation assays were conducted in HEK293T cells. Data were analyzed using analysis of variance or Student t test. RESULTS: Compared with ND, HFD increased the mRNA expressions of gluconeogenesis genes (2.40-fold to 2.60-fold) (P < 0.05) and reduced protein kinase B (AKT) phosphorylation levels (0.28-fold to 0.34-fold) (P < 0.05) in croakers. However, inhibition of SREBP1 by fatostatin addition or SREBP1 knockdown reduced the mRNA expressions of gluconeogenesis genes (P < 0.05) and increased AKT phosphorylation levels (P < 0.05) in hepatocytes, compared with that by PA treatment. Moreover, fatostatin addition or SREBP1 knockdown also increased the mRNA expressions of irs1 (P < 0.05) and reduced serine phosphorylation of IRS1 (P < 0.05). Furthermore, SREBP1 inhibited IRS1 transcriptions by binding to its promoter and induced IRS1 serine phosphorylation by activating diacylglycerol-protein kinase Cε signaling. CONCLUSIONS: This study reveals the role of SREBP1 in hepatic gluconeogenesis under HFD condition in croakers, which may provide a potential strategy for improving HFD-induced glucose intolerance.

Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation.

Trabecular meshwork (TM) cells are contractile and mechanosensitive, and they aid in maintaining intraocular pressure (IOP) homeostasis. Lipids are attributed to modulating TM contractility, with poor mechanistic understanding. In this study using human TM cells, we identify the mechanosensing role of the transcription factors sterol regulatory element binding proteins (SREBPs) involved in lipogenesis. By constitutively activating SREBPs and pharmacologically inactivating SREBPs, we have mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo, respectively, results in significant IOP lowering. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways as well as the levels of the phospholipid, cholesterol, and triglyceride. Further, we show that fatostatin mitigated actin polymerization machinery and stabilization, and decreased ECM synthesis and secretion. We thus postulate that lowering lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics.