Cut from the same cloth: RNAs transcribed from regulatory elements.

A certain degree of chromatin openness is necessary for the activity of transcription-regulating regions within the genome, facilitating accessibility to RNA polymerases and subsequent synthesis of regulatory element RNAs (regRNAs) from these regions. The rapidly increasing number of studies underscores the significance of regRNAs across diverse cellular processes and diseases, challenging the paradigm that these transcripts are non-functional transcriptional noise. This review explores the multifaceted roles of regRNAs in human cells, encompassing rather well-studied entities such as promoter RNAs and enhancer RNAs (eRNAs), while also providing insights into overshadowed silencer RNAs and insulator RNAs. Furthermore, we assess notable examples of shorter regRNAs, like miRNAs, snRNAs, and snoRNAs, playing important roles. Expanding our discourse, we deliberate on the potential usage of regRNAs as biomarkers and novel targets for cancer and other human diseases.

Transcriptional Regulation of the Lineage-Determining Gene PU.1 in Normal and Malignant Hematopoiesis: Current Understanding and Therapeutic Perspective.

The ETS transcription factor PU.1 plays an essential role in blood cell development. Its precise expression pattern is governed by cis-regulatory elements (CRE) acting at the chromatin level. CREs mediate the fine-tuning of graded levels of PU.1, deviations of which can cause acute myeloid leukemia. In this review, we perform an in-depth analysis of the regulation of PU.1 expression in normal and malignant hematopoiesis. We elaborate on the role of trans-acting factors and the biomolecular interplays in mediating local chromatin dynamics. Moreover, we discuss the current understanding of CRE bifunctionality exhibiting enhancer or silencer activities in different blood cell lineages and future directions toward gene-specific chromatin-targeted therapeutic development.

KSHV genome harbors both constitutive and lytically induced enhancers.

Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements, such as enhancers and silencers, can regulate gene expression in a position- and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses.IMPORTANCEIn this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA to regulate latency to lytic transition.

Surface PD-1 expression in T cells is suppressed by HNRNPK through an exonic splicing silencer on exon 3.

OBJECTIVE: Immunotherapy targeting programmed cell death 1 (PDCD1 or PD-1) and its ligands has shown remarkable promise and the regulation mechanism of PD-1 expression has received arising attention in recent years. PDCD1 exon 3 encodes the transmembrane domain and the deletion of exon 3 produces a soluble protein isoform of PD-1 (sPD-1), which can enhance immune response by competing with full-length PD-1 protein (flPD-1 or surface PD-1) on T cell surface. However, the mechanism of PDCD1 exon 3 skipping is unclear. METHODS: The online SpliceAid program and minigene expression system were used to analyze potential splicing factors involved in the splicing event of PDCD1 exon 3. The potential binding motifs of heterogeneous nuclear ribonucleoprotein K (HNRNPK) on exon 3 predicted by SpliceAid were mutated by site-directed mutagenesis technology, which were further verified by pulldown assay. Antisense oligonucleotides (ASOs) targeting the exonic splicing silencer (ESS) on PDCD1 exon 3 were synthesized and screened to suppress the skipping of exon 3. The alternative splicing of PDCD1 exon 3 was analyzed by semiquantitative reverse transcription PCR. Western blot and flow cytometry were performed to detect the surface PD-1 expression in T cells. RESULTS: HNRNPK was screened as a key splicing factor that promoted PDCD1 exon 3 skipping, causing a decrease in flPD-1 expression on T cell membrane and an increase in sPD-1 expression. Mechanically, a key ESS has been identified on exon 3 and can be bound by HNRNPK protein to promote exon 3 skipping. Blocking the interaction between ESS and HNRNPK with an ASO significantly reduced exon 3 skipping. Importantly, HNRNPK can promote exon 3 skipping of mouse Pdcd1 gene as well. CONCLUSIONS: Our study revealed a novel evolutionarily conserved regulatory mechanism of PD-1 expression. The splicing factor HNRNPK markedly promoted PDCD1 exon 3 skipping by binding to the ESS on PDCD1 exon 3, resulting in decreased expression of flPD-1 and increased expression of sPD-1 in T cells.

Quantification of suppressor effects on breech and action noise from AR-15 pattern firearms and its implications for the protection of human hearing.

Noise from firearms is well known to be harmful to human hearing. This problem has been addressed by various military units through the use of muzzle suppressors. However, as suppressor technology has advanced, shooters report hearing the mechanical action of gas-operated semi-automatic rifles (ArmaLite Rifle Model 15 style aka AR-15) as being louder than the suppressed muzzle noise. This study aims to evaluate if harmful noise is present in the shooter's ear, even when impulse noise emanating from the muzzle is suppressed. To characterize the impulse noise of the firearm action caused by the reciprocation of the bolt carrier group (BCG) and subsequent impact when it returns to battery (the forward locked position), the muzzle of a rifle was placed through a constructed plywood wall, and the noise of the action/breech was measured independently from the muzzle noise. This research finds that the impact of the BCG returning to battery (132 dBZ) has the potential to be harmful to the shooter's hearing even when the noise from the muzzle is effectively suppressed.

Evolution of a Human-Specific De Novo Open Reading Frame and Its Linked Transcriptional Silencer.

In the human genome, two short open reading frames (ORFs) separated by a transcriptional silencer and a small intervening sequence stem from the gene SMIM45. The two ORFs show different translational characteristics, and they also show divergent patterns of evolutionary development. The studies presented here describe the evolution of the components of SMIM45. One ORF consists of an ultra-conserved 68 amino acid (aa) sequence, whose origins can be traced beyond the evolutionary age of divergence of the elephant shark, ~462 MYA. The silencer also has ancient origins, but it has a complex and divergent pattern of evolutionary formation, as it overlaps both at the 68 aa ORF and the intervening sequence. The other ORF consists of 107 aa. It develops during primate evolution but is found to originate de novo from an ancestral non-coding genomic region with root origins within the Afrothere clade of placental mammals, whose evolutionary age of divergence is ~99 MYA. The formation of the complete 107 aa ORF during primate evolution is outlined, whereby sequence development is found to occur through biased mutations, with disruptive random mutations that also occur but lead to a dead-end. The 107 aa ORF is of particular significance, as there is evidence to suggest it is a protein that may function in human brain development. Its evolutionary formation presents a view of a human-specific ORF and its linked silencer that were predetermined in non-primate ancestral species. The genomic position of the silencer offers interesting possibilities for the regulation of transcription of the 107 aa ORF. A hypothesis is presented with respect to possible spatiotemporal expression of the 107 aa ORF in embryonic tissues.

Compressor pulsation noise attenuation using reactive silencer with various configurations: A theoretical and experimental study.

Compressors are a significant source of noise in various industries. Silencers can be utilized to mitigate this noise. This study aims to design and construct an expansion silencer that can effectively reduce the pulsating noise produced by a reciprocating compressor. This study employed a model-experimental approach to investigate the performance of four different sizes of expansion silencers in controlling the pulsating noise in the suction part of the compressor. Initially, the silencers' sound transmission loss and pressure loss were simulated using the finite element method with COMSOL software. Subsequently, the sound transmission loss of the silencers was measured according to the E261109 standard using an impedance tube. Finally, the pressure loss of the silencers was measured using a Pitot tube upstream and downstream of the silencer at various flow rates. The results of the modeling showed that increasing the diameter of the silencer leads to an increase in transmission loss at all frequencies. Additionally, raising the length of the silencer only increased the number of sound transmission loss peaks in the frequency bandwidth without significant change in sound transmission loss. Furthermore, the results of the experimental measurements with an impedance tube revealed that increasing the diameter results in increased transmission loss, while increasing the silencer length leads to an increase in the number of transmission loss peaks without altering the transmission loss. Moreover, the modeling and experimental pressure loss results indicated that increasing the diameter of the expansion chamber causes an increase in pressure loss, while increasing the length of the expansion chamber results in a minor change in pressure loss. Finally, the research results showed relatively good agreement between modeling and experimental outcomes.

A screen for regeneration-associated silencer regulatory elements in zebrafish.

Regeneration involves gene expression changes explained in part by context-dependent recruitment of transcriptional activators to distal enhancers. Silencers that engage repressive transcriptional complexes are less studied than enhancers and more technically challenging to validate, but they potentially have profound biological importance for regeneration. Here, we identified candidate silencers through a screening process that examined the ability of DNA sequences to limit injury-induced gene expression in larval zebrafish after fin amputation. A short sequence (s1) on chromosome 5 near several genes that reduce expression during adult fin regeneration could suppress promoter activity in stable transgenic lines and diminish nearby gene expression in knockin lines. High-resolution analysis of chromatin organization identified physical associations of s1 with gene promoters occurring preferentially during fin regeneration, and genomic deletion of s1 elevated the expression of these genes after fin amputation. Our study provides methods to identify "tissue regeneration silencer elements" (TRSEs) with the potential to reduce unnecessary or deleterious gene expression during regeneration.

Erythropoietin-overexpressed umbilical cord mesenchymal stem cells inhibit neuroapoptosis in ischemic-hypoxic SH-SY5Y and its mechanism / 中国组织工程研究

BACKGROUND:Previous studies have successfully constructed erythropoietin-overexpressed umbilical cord mesenchymal stem cells.It was found that the apoptosis of ischemic and hypoxic human neuroblastoma cell line(SH-SY5Y)was significantly reduced by erythropoietin-overexpressed umbilical cord mesenchymal stem cells. OBJECTIVE:To explore the possible neuroprotective mechanisms of erythropoietin-overexpressed umbilical cord mesenchymal stem cells against ischemic-hypoxic SH-SY5Y and their associated epigenetic mechanisms. METHODS:Oxygen-glucose deprivation was applied to ischemia-hypoxia-induced SH-SY5Y cell injury,and multifactorial assays were applied to detect the expression levels of inflammatory factors in the cells before and after hypoxia and co-culture,respectively,with mesenchymal stem cells,as well as lentiviral-transfected null-loaded plasmids of the negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells.The expression levels of supernatant inflammatory factors were detected by multifactor assay after co-culture.Proteomics was used to detect the differentially expressed proteins of negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells.Cleavage under targets and tagmentation sequencing was applied to detect genomic H3K4me2 modification,and joint analysis was conducted with RNA-sequencing.Lentiviral vector infection was applied to construct the stable knockdown of REST in SH-SY5Y cells.qRT-PCR and western blot assay were performed to detect the expression level of REST.The apoptosis was detected by flow cytometry after co-culture of oxygen-glucose deprivation treatment with erythropoietin-overexpressed umbilical cord mesenchymal stem cells.The expression difference of H3K36me3 group proteins was detected by western blot assay,and transcriptome sequencing was performed to analyze the differentially expressed genes. RESULTS AND CONCLUSION:(1)Compared with the control group,monocyte chemotactic protein 1,interleukin-6,interleukin-18,and interleukin-1 beta,interferon α2,and interleukin-23 levels significantly increased in the cerebrospinal fluid supernatant of patients with ischemic-hypoxic encephalopathy(P<0.01).(2)After co-culturing SH-SY5Y cells with erythropoietin-overexpressed umbilical cord mesenchymal stem cells under ischemia and hypoxia,the expression levels of monocyte chemotactic protein 1 and interleukin-6 were significantly reduced.(3)Analysis of protein network interactions revealed significant downregulation of monocyte chemotactic protein 1,interleukin-6 related regulatory proteins CXCL1 and BGN.(4)Transcriptome sequencing analysis found that pro-inflammatory genes were down-regulated,and functional enrichment of histone modifications,and the expression of transcription factors REST and TET3 significantly up-regulated in the erythropoietin-overexpressed umbilical cord mesenchymal stem cell group compared with the negative control mesenchymal stem cell group.(5)Combined analysis of transcriptome sequencing and cleavage under targets and tagmentation revealed changes in epigenetic levels as well as significant activation of the promoter regions of transcription factors REST and TET3.(6)Stable knockdown REST in SH-SY5Y cells was successfully constructed;the transcript levels of REST mRNA and protein expression were both decreased.(7)After the REST knockdown SH-SY5Y cells were co-cultured with erythropoietin-overexpressed umbilical cord mesenchymal stem cells,apoptosis was significantly increased and H3K36me3 expression was significantly decreased.Transcriptome sequencing results showed that the expression of inflammation-related genes Aldh1l2 and Cth,as well as apoptosis-suppressor genes Mapk8ip1 and Sod2 was reduced at mRNA transcription level(P<0.01).(8)It is concluded that erythropoietin-overexpressed umbilical cord mesenchymal stem cells activated the expression of REST and TET3 by altering the kurtosis of H3K4me2 and upregulated the modification level of H3K36me3,which in turn regulated the expression of inflammation-related genes Aldh1l2 and Cth,as well as apoptosis-suppressor genes Mapk8ip1 and Sod2,and facilitated neuronal survival.

Conditional knockout of REST/NRSF in excitatory neurons reduces seizure susceptibility to chemical kindling.

The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is an epigenetic master regulator that plays a crucial role during nervous system development and maturation. REST function was originally described during development, where it determines neuronal phenotype. However, recent studies showed that REST participates in several processes in the adult brain, including neuronal plasticity and epileptogenesis. In this regard, the relationships between REST and epilepsy are still controversial and need further investigation. As forebrain excitatory neurons are the common final pathway of seizure susceptibility, we investigated the role of REST in epilepsy by inducing REST conditional knockout (REST-cKO) specifically in excitatory neurons of the hippocampus. To target the excitatory neuronal population, we cloned the calcium/calmodulin-dependent protein kinase IIα minimal promoter upstream of Cre recombinase. After assessing the specificity of the promoter's expression, the transgenes were packaged in an engineered adeno-associated virus able to cross the blood-brain and blood-cerebrospinal fluid barriers and delivered in the lateral ventricles of 2-month-old RESTflox/flox mice to characterize, after 1 month, the cognitive phenotype and the seizure propensity. We show that REST-cKO mice display lower levels of anxiety in the light-dark test with respect to control mice but have unaltered motor, social, and cognitive profiles. The evaluation of the susceptibility to epileptic seizures showed that REST-cKO mice are more resistant to pentylenetetrazole-induced kindling but not to seizures induced by a single administration of the convulsant and show higher survival rates. Overall, these data suggest that the absence of REST in forebrain excitatory neurons decreases seizure susceptibility, pointing to a pro-epileptogenic role of the transcriptional repressor under conditions of pathological excitation/inhibition imbalance.

Brucella MucR acts as an H-NS-like protein to silence virulence genes and structure the nucleoid.

Histone-like nucleoid structuring (H-NS) and H-NS-like proteins serve as global gene silencers and work with antagonistic transcriptional activators (counter-silencers) to properly coordinate the expression of virulence genes in pathogenic bacteria. In Brucella, MucR has been proposed as a novel H-NS-like gene silencer, but direct experimental evidence is lacking. Here, we show that MucR serves as an H-NS-like silencer of the Brucella abortus genes encoding the polar autotransporter adhesins BtaE and BmaC, the c-di-GMP-specific phosphodiesterase BpdB, and the quorum-sensing regulator BabR. We also demonstrate that the MarR-type transcriptional activator MdrA can displace MucR from the btaE promoter, supporting the existence of MucR counter-silencers in Brucella. Moreover, our chromatin immunoprecipitation (ChIP)-seq analysis identified 546 MucR enrichment peaks along the genome, including in the promoters of the genes encoding the Type IV secretion machinery and effectors and the quorum-sensing regulator VjbR. Importantly, MucR ChIP-seq peaks overlap with the previously described binding sites for the transcriptional activators VjbR, BvrR, and CtrA suggesting that these regulators serve as MucR counter-silencers and work in concert with MucR to coordinate virulence gene expression in Brucella. In addition, using chromosome conformation capture (Hi-C), we show that like H-NS in Escherichia coli, MucR alters the global structure of the Brucella nucleoid. Finally, a copy of the E. coli hns rescues the distinctive growth defect and elevated btaE expression of a B. abortus mucR mutant. Together, these findings solidify the role of MucR as a novel type of H-NS-like protein and suggest that MucR's gene-silencing properties play a key role in virulence in Brucella. IMPORTANCE Histone-like nucleoid structuring (H-NS) and H-NS-like proteins coordinate host-associated behaviors in many pathogenic bacteria, often through forming silencer/counter-silencer pairs with signal-responsive transcriptional activators to tightly control gene expression. Brucella and related bacteria do not encode H-NS or homologs of known H-NS-like proteins, and it is unclear if they have other proteins that perform analogous functions during pathogenesis. In this work, we provide compelling evidence for the role of MucR as a novel H-NS-like protein in Brucella. We show that MucR possesses many of the known functions attributed to H-NS and H-NS-like proteins, including the formation of silencer/counter-silencer pairs to control virulence gene expression and global structuring of the nucleoid. These results uncover a new role for MucR as a nucleoid structuring protein and support the importance of temporal control of gene expression in Brucella and related bacteria.

DeepICSH: a complex deep learning framework for identifying cell-specific silencers and their strength from the human genome.

Silencers are noncoding DNA sequence fragments located on the genome that suppress gene expression. The variation of silencers in specific cells is closely related to gene expression and cancer development. Computational approaches that exclusively rely on DNA sequence information for silencer identification fail to account for the cell specificity of silencers, resulting in diminished accuracy. Despite the discovery of several transcription factors and epigenetic modifications associated with silencers on the genome, there is still no definitive biological signal or combination thereof to fully characterize silencers, posing challenges in selecting suitable biological signals for their identification. Therefore, we propose a sophisticated deep learning framework called DeepICSH, which is based on multiple biological data sources. Specifically, DeepICSH leverages a deep convolutional neural network to automatically capture biologically relevant signal combinations strongly associated with silencers, originating from a diverse array of biological signals. Furthermore, the utilization of attention mechanisms facilitates the scoring and visualization of these signal combinations, whereas the employment of skip connections facilitates the fusion of multilevel sequence features and signal combinations, thereby empowering the accurate identification of silencers within specific cells. Extensive experiments on HepG2 and K562 cell line data sets demonstrate that DeepICSH outperforms state-of-the-art methods in silencer identification. Notably, we introduce for the first time a deep learning framework based on multi-omics data for classifying strong and weak silencers, achieving favorable performance. In conclusion, DeepICSH shows great promise for advancing the study and analysis of silencers in complex diseases. The source code is available at https://github.com/lyli1013/DeepICSH.

Central nervous system involvement in two siblings affected by hereditary transthyretin amyloidosis 30 years after liver transplantation: a model for gene-silencing therapies.

Hereditary transthyretin amyloidosis (ATTRv) is a genetic, autosomal dominant, severe disease characterized by progressive sensory-motor polyneuropathy, cardiomyopathy, dysautonomia, renal and eyes involvement, provoked by the deposition of the mutated and unstable transthyretin protein. In past decades, liver transplant, avoiding the synthesis of the pathologic protein, has been a good, even if not resolutive, treatment. In this report we describe two siblings affected with ATTRv, who developed first symptoms of disease at a young age and underwent a liver transplant with prompt resolution of clinical manifestations. After several years, central nervous system and eyes symptoms relapsed despite treatment, considering that the synthesis of mutated protein continues in choroid plexus, a locum where current therapies are unable to act. In our opinion, these cases represent a long-term prognostic model for the novel gene-silencers approved for ATTRv, because they share a similar therapeutic effect with liver transplant: the block of mutated protein synthesis limited only in the main transthyretin (TTR) production organ is able to prevent the progression of disease only for some years, but not to avoid long-term clinical worsening due to extra-hepatic production of TTR. Novel future therapeutic strategies are demanded to guarantee a better long-term stabilization of symptomatology.

Haplotype mapping of H3K27me3-associated chromatin interactions defines topological regulation of gene silencing in rice.

Histone modification H3K27me3 is an important chromatin mark that plays vital roles in repressing expression of developmental genes. Here, we construct high-resolution 3D genome maps using long-read chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and characterize H3K27me3-associated chromatin interactions in an elite rice hybrid, Shanyou 63. We find that many H3K27me3-marked regions may function as silencer-like regulatory elements. The silencer-like elements can come into proximity with distal target genes via forming chromatin loops in 3D space of the nuclei, regulating gene silencing and plant traits. Natural and induced deletion of silencers upregulate expression of distal connected genes. Furthermore, we identify extensive allele-specific chromatin loops. We find that genetic variations alter allelic chromatin topology, thus modulating allelic gene imprinting in rice hybrids. In conclusion, the characterization of silencer-like regulatory elements and haplotype-resolved chromatin interaction maps provide insights into the understanding of molecular mechanisms underlying allelic gene silencing and plant trait controlling.

Whole-genome functional characterization of RE1 silencers using a modified massively parallel reporter assay.

Both upregulation and downregulation by cis-regulatory elements help modulate precise gene expression. However, our understanding of repressive elements is far more limited than activating elements. To address this gap, we characterized RE1, a group of transcriptional silencers bound by REST, at genome-wide scale using a modified massively parallel reporter assay (MPRAduo). MPRAduo empirically defined a minimal binding strength of REST (REST motif-intrinsic value [m-value]), above which cofactors colocalize and silence transcription. We identified 1,500 human variants that alter RE1 silencing and found that their effect sizes are predictable when they overlap with REST-binding sites above the m-value. Additionally, we demonstrate that non-canonical REST-binding motifs exhibit silencer function only if they precisely align half sites with specific spacer lengths. Our results show mechanistic insights into RE1, which allow us to predict its activity and effect of variants on RE1, providing a paradigm for performing genome-wide functional characterization of transcription-factor-binding sites.

Electron microscopy reveals toroidal shape of master neuronal cell differentiator REST - RE1-silencing transcription factor.

The RE1-Silencing Transcription factor (REST) is essential for neuronal differentiation. Here, we report the first 18.5-angstrom electron microscopy structure of human REST. The refined electron map suggests that REST forms a torus that can accommodate DNA double-helix in the central hole. Additionally, we quantitatively described REST binding to the canonical DNA sequence of the neuron-restrictive silencer element. We developed protocols for the expression and purification of full-length REST and the shortened variant REST-N62 produced by alternative splicing. We tested the mutual interaction of full-length REST and the splicing variant REST-N62. Revealed structure-function relationships of master neuronal repressor REST will allow finding new biological ways of prevention and treatment of neurodegenerative disorders and diseases.

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.

Activating and repressing gene expression between chromosomes during stochastic fate specification.

DNA elements act across long genomic distances to regulate gene expression. During transvection in Drosophila, DNA elements on one allele of a gene act between chromosomes to regulate expression of the other allele. Little is known about the biological roles and developmental regulation of transvection. Here, we study the stochastic expression of spineless (ss) in photoreceptors in the fly eye to understand transvection. We determine a biological role for transvection in regulating expression of naturally occurring ss alleles. We identify DNA elements required for activating and repressing transvection. Different enhancers participate in transvection at different times during development to promote gene expression and specify cell fates. Bringing a silencer element on a heterologous chromosome into proximity with the ss locus "reconstitutes" the gene, leading to repression. Our studies show that transvection regulates gene expression via distinct DNA elements at specific timepoints in development, with implications for genome organization and architecture.

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).

Novel Small Molecule Positive Allosteric Modulator SPAM1 Triggers the Nuclear Translocation of PAC1-R to Exert Neuroprotective Effects through Neuron-Restrictive Silencer Factor.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) exerts effective neuroprotective activity through its specific receptor, PAC1-R. We accidentally discovered that as a positive allosteric modulator (PAM) of PAC1-R, the small-molecule PAM (SPAM1) has a hydrazide-like structure, but different binding characteristics, from hydrazide for the N-terminal extracellular domain of PAC1-R (PAC1-R-EC1). SPAM1 had a significant neuroprotective effect against oxidative stress, both in a cell model treated with hydrogen peroxide (H2O2) and an aging mouse model induced by D-galactose (D-gal). SPAM1 was found to block the decrease in PACAP levels in brain tissues induced by D-gal and significantly induced the nuclear translocation of PAC1-R in PAC1R-CHO cells and mouse retinal ganglion cells. Nuclear PAC1-R was subjected to fragmentation and the nuclear 35 kDa, but not the 15 kDa fragments, of PAC1-R interacted with SP1 to upregulate the expression of Huntingtin (Htt), which then exerted a neuroprotective effect by attenuating the binding availability of the neuron-restrictive silencer factor (NRSF) to the neuron-restrictive silencer element (NRSE). This resulted in an upregulation of the expression of NRSF-related neuropeptides, including PACAP, the brain-derived neurotrophic factor (BDNF), tyrosine hydroxylase (TH), and synapsin-1 (SYN1). The novel mechanism reported in this study indicates that SPAM1 has potential use as a drug, as it exerts a neuroprotective effect by regulating NRSF.