Gene Regulatory Sites Research Articles

Deciphering the dynamics: Exploring the impact of mechanical forces on histone acetylation.

Living cells navigate a complex landscape of mechanical cues that influence their behavior and fate, originating from both internal and external sources. At the molecular level, the translation of these physical stimuli into cellular responses relies on the intricate coordination of mechanosensors and transducers, ultimately impacting chromatin compaction and gene expression. Notably, epigenetic modifications on histone tails govern the accessibility of gene-regulatory sites, thereby regulating gene expression. Among these modifications, histone acetylation emerges as particularly responsive to the mechanical microenvironment, exerting significant control over cellular activities. However, the precise role of histone acetylation in mechanosensing and transduction remains elusive due to the complexity of the acetylation network. To address this gap, our aim is to systematically explore the key regulators of histone acetylation and their multifaceted roles in response to biomechanical stimuli. In this review, we initially introduce the ubiquitous force experienced by cells and then explore the dynamic alterations in histone acetylation and its associated co-factors, including HDACs, HATs, and acetyl-CoA, in response to these biomechanical cues. Furthermore, we delve into the intricate interactions between histone acetylation and mechanosensors/mechanotransducers, offering a comprehensive analysis. Ultimately, this review aims to provide a holistic understanding of the nuanced interplay between histone acetylation and mechanical forces within an academic framework.

The role of DNA demethylation in liver to pancreas transdifferentiation

BackgroundInsulin producing cells generated by liver cell transdifferentiation, could serve as an attractive source for regenerative medicine. The present study assesses the relationship between DNA methylation pTFs induced liver to pancreas transdifferentiation.ResultsThe transdifferentiation process is associated with DNA demethylation, mainly at gene regulatory sites, and with increased expression of these genes. Active inhibition of DNA methylation promotes the pancreatic transcription factor-induced transdifferentiation process, supporting a causal role for DNA demethylation in this process.ConclusionsTransdifferentiation is associated with global DNA hypomethylation, and with increased expression of specific demethylated genes. A combination of epigenetic modulators may be used to increase chromatin accessibility of the pancreatic transcription factors, thus promoting the efficiency of the developmental process.

Comprehensive characterization of tissue-specific chromatin accessibility in L2 Caenorhabditis elegans nematodes

Recently developed single-cell technologies allow researchers to characterize cell states at ever greater resolution and scale. Caenorhabditis elegans is a particularly tractable system for studying development, and recent single-cell RNA-seq studies characterized the gene expression patterns for nearly every cell type in the embryo and at the second larval stage (L2). Gene expression patterns give insight about gene function and into the biochemical state of different cell types; recent advances in other single-cell genomics technologies can now also characterize the regulatory context of the genome that gives rise to these gene expression levels at a single-cell resolution. To explore the regulatory DNA of individual cell types in C. elegans, we collected single-cell chromatin accessibility data using the sci-ATAC-seq assay in L2 larvae to match the available single-cell RNA-seq data set. By using a novel implementation of the latent Dirichlet allocation algorithm, we identify 37 clusters of cells that correspond to different cell types in the worm, providing new maps of putative cell type–specific gene regulatory sites, with promise for better understanding of cellular differentiation and gene regulation.

Nuclear receptor SHP dampens transcription function and abrogates mitotic chromatin association of PXR and ERα via intermolecular interactions

Mitosis is a cellular process that produces two identical progenies. Genome-wide transcription is believed to be silenced during mitosis. However, some transcription factors have been reported to associate with the mitotic chromatin to uphold a role in ‘gene-bookmarking’. Here, we investigated the dynamic role of nuclear receptor SHP during cell cycle, and observed intermolecular interactions with PXR and ERα. This was reflected in altered subcellular localization, transcription function and mitotic chromatin behavior of these receptors. Subsequently, by in silico and live cell imaging approaches we identified the minimal domain(s) and crucial amino-acid residues required for such receptor-receptor interactions. It was apparent that both PXR/ERα interact with SHP to translocate cytoplasmic RFP-tagged SHP into the nucleus. In addition, during mitosis SHP interacted with some of the key nuclear receptors, altering partners, as well as, its own relationship with mitotic chromatin. SHP displaced a major fraction of PXR and ERα from the mitotic chromatin while promoted its own weak association reflected in its binding. Since SHP lacks DBD this association is attributed to receptor-receptor interactions rather than SHP-DNA interactions. The abrogation of PXR and ERα from the mitotic chromatin by SHP implies potential implications in regulation of gene bookmarking events in cellular development. Overall, it is concluded that intermolecular interactions between SHP and partner PXR/ERα result in attenuation of target promoter activities. It is proposed that SHP may act as an indirect physiological regulator and functions in a hog-tie manner by displacing the interacting transcription factor from gene regulatory sites.

Tumor necrosis factor (TNF)-α- 308 G/A gene polymorphism (rs1800629) in Egyptian patients with alopecia areata and vitiligo, a laboratory and in silico analysis.

Purpose & methodsSeveral single-nucleotide polymorphisms (SNPs) in the promoter region of the TNF-α gene can cause variations in the gene regulatory sites and act as risk factors for some autoimmune disorders as alopecia areata (AA) and vitiligo. This study aimed to detect the serum TNF-α (sTNF) level (by ELISA) and the rs1800629 (by real-time PCR) among AA and vitiligo Egyptian patients and to determine their relation with disease duration and severity. In silico analysis of this SNP to study the molecular regulation of the mutant genotypes was also done.ResultsIn AA patients, no risk was associated with the mutant genotypes vs. the normal genotype, or with A allele vs. G allele. The risk of vitiligo was significantly higher with the G/A and A/A genotypes compared with HCs (p = 0.011). Similarly, a significantly increased risk was noted in patients with A allele vs. G allele (p<0.0001). In AA and vitiligo patients, a significant increase in sTNF-α levels was noted in the mutant G/A genotypes vs. the normal G/G genotype (p<0.0001) and in the A allele vs the G allele (p<0.0001). According to the in silico analysis, this SNP could mainly affect the SP1 transcription factor binding site with subsequent effect on TNF-α expression.ConclusionAccording to results of the laboratory and the in silico study, the mutant TNF-α (308) genotypes were risk factors that conferred susceptibility to vitiligo among Egyptian patients but had no effect on the susceptibility to AA.

The chromatin remodeling enzyme Chd4 regulates genome architecture in the mouse brain

The development and function of the brain require tight control of gene expression. Genome architecture is thought to play a critical regulatory role in gene expression, but the mechanisms governing genome architecture in the brain in vivo remain poorly understood. Here, we report that conditional knockout of the chromatin remodeling enzyme Chd4 in granule neurons of the mouse cerebellum increases accessibility of gene regulatory sites genome-wide in vivo. Conditional knockout of Chd4 promotes recruitment of the architectural protein complex cohesin preferentially to gene enhancers in granule neurons in vivo. Importantly, in vivo profiling of genome architecture reveals that conditional knockout of Chd4 strengthens interactions among developmentally repressed contact domains as well as genomic loops in a manner that tightly correlates with increased accessibility, enhancer activity, and cohesin occupancy at these sites. Collectively, our findings define a role for chromatin remodeling in the control of genome architecture organization in the mammalian brain.

Binding of an X-Specific Condensin Correlates with a Reduction in Active Histone Modifications at Gene Regulatory Elements.

Condensins are evolutionarily conserved protein complexes that are required for chromosome segregation during cell division and genome organization during interphase. In Caenorhabditis elegans, a specialized condensin, which forms the core of the dosage compensation complex (DCC), binds to and represses X chromosome transcription. Here, we analyzed DCC localization and the effect of DCC depletion on histone modifications, transcription factor binding, and gene expression using chromatin immunoprecipitation sequencing and mRNA sequencing. Across the X, the DCC accumulates at accessible gene regulatory sites in active chromatin and not heterochromatin. The DCC is required for reducing the levels of activating histone modifications, including H3K4me3 and H3K27ac, but not repressive modification H3K9me3. In X-to-autosome fusion chromosomes, DCC spreading into the autosomal sequences locally reduces gene expression, thus establishing a direct link between DCC binding and repression. Together, our results indicate that DCC-mediated transcription repression is associated with a reduction in the activity of X chromosomal gene regulatory elements.

BayesPI-BAR2: A New Python Package for Predicting Functional Non-coding Mutations in Cancer Patient Cohorts.

Most of somatic mutations in cancer occur outside of gene coding regions. These mutations may disrupt the gene regulation by affecting protein-DNA interaction. A study of these disruptions is important in understanding tumorigenesis. However, current computational tools process DNA sequence variants individually, when predicting the effect on protein-DNA binding. Thus, it is a daunting task to identify functional regulatory disturbances among thousands of mutations in a patient. Previously, we have reported and validated a pipeline for identifying functional non-coding somatic mutations in cancer patient cohorts, by integrating diverse information such as gene expression, spatial distribution of the mutations, and a biophysical model for estimating protein binding affinity. Here, we present a new user-friendly Python package BayesPI-BAR2 based on the proposed pipeline for integrative whole-genome sequence analysis. This may be the first prediction package that considers information from both multiple mutations and multiple patients. It is evaluated in follicular lymphoma and skin cancer patients, by focusing on sequence variants in gene promoter regions. BayesPI-BAR2 is a useful tool for predicting functional non-coding mutations in whole genome sequencing data: it allows identification of novel transcription factors (TFs) whose binding is altered by non-coding mutations in cancer. BayesPI-BAR2 program can analyze multiple datasets of genome-wide mutations at once and generate concise, easily interpretable reports for potentially affected gene regulatory sites. The package is freely available at http://folk.uio.no/junbaiw/BayesPI-BAR2/.

Reduction of multiscale stochastic biochemical reaction networks using exact moment derivation.

Biochemical reaction networks (BRNs) in a cell frequently consist of reactions with disparate timescales. The stochastic simulations of such multiscale BRNs are prohibitively slow due to high computational cost for the simulations of fast reactions. One way to resolve this problem uses the fact that fast species regulated by fast reactions quickly equilibrate to their stationary distribution while slow species are unlikely to be changed. Thus, on a slow timescale, fast species can be replaced by their quasi-steady state (QSS): their stationary conditional expectation values for given slow species. As the QSS are determined solely by the state of slow species, such replacement leads to a reduced model, where fast species are eliminated. However, it is challenging to derive the QSS in the presence of nonlinear reactions. While various approximation schemes for the QSS have been developed, they often lead to considerable errors. Here, we propose two classes of multiscale BRNs which can be reduced by deriving an exact QSS rather than approximations. Specifically, if fast species constitute either a feedforward network or a complex balanced network, the reduced model based on the exact QSS can be derived. Such BRNs are frequently observed in a cell as the feedforward network is one of fundamental motifs of gene or protein regulatory networks. Furthermore, complex balanced networks also include various types of fast reversible bindings such as bindings between transcriptional factors and gene regulatory sites. The reduced models based on exact QSS, which can be calculated by the computational packages provided in this work, accurately approximate the slow scale dynamics of the original full model with much lower computational cost.

Transcriptional coregulator RIP140: an essential regulator of physiology.

Transcriptional coregulators drive gene regulatory decisions in the transcriptional space. Although transcription factors including all nuclear receptors provide a docking platform for coregulators to bind, these proteins bring enzymatic capabilities to the gene regulatory sites. RIP140 is a transcriptional coregulator essential for several physiological processes, and aberrations in its function may lead to diseased states. Unlike several other coregulators that are known either for their coactivating or corepressing roles, in gene regulation, RIP140 is capable of acting both as a coactivator and a corepressor. The role of RIP140 in female reproductive axis and recent findings of its role in carcinogenesis and adipose biology have been summarised.

Scientists Explore a Master Genetic Switch That Plays a Key Role in Energy Metabolism and Human Brain Evolution and Function

Scientists have long used comparative animal studies to better understand the nuances of human evolution, from making diverse body plans to the emergence of entirely powerful and unique features structures, including the human brain. Now, in a new study appearing in the advanced online edition of Molecular Biology and Evolution, corresponding authors Perdomo-Sabogal et al. (2016) have explored the global gene regulator GABPa to better understand its influence as a master switch. Their work honed in on human-specific DNA regions potentially contributing to human evolution in key functions including cell energy, division and death, and diseases ranging from brain disorders to diabetes. Many of the GABPa gene regulatory sites were found to be significantly enriched at genes important for brain, central nervous system and spinal cord functions that are unique to humans, an area ripe for future research, and GABPa deficits linked to Alzheimer’s and Parkinson diseases, breast cancer, and autism. GABPa exerts its profound genomic influence by a tandem repeat of four highly conserved DNA letters. The GGAA consensus motif is found to control genes in all primates, mice, dogs, and cows, underscoring its importance in these animals. By performing ChIP-Seq experiments in a human cell line, the group found that GABPa exercises its influence across the genome at 11,619 putative GABPa binding sites representing nearly 4,000 genes. Through a tour de force sequence comparison of the human GABPa binding sites with similar sequences from 34 mammals, they identified 224 GABPa binding sites unique to humans. Comparing chimpanzee, macaque, and human cell lines and mutating the GGAA sequence to more ancestral states to determine how these changes affected GABPa function, they further narrowed down their function. For instance, when the human GABPa binding sites were introduced into primate cells, gene expression was increased up to five times in 17 out of 18 cases, indicating that a recent human mutation in GABPa resulted in higher gene expression and forces behind the evolution of novel, uniquely human functions. “Mutations that cause changes in the regulation of gene activities are one of the major factors in shaping species during evolution,” said corresponding author Robert Querfurth. “Our study demonstrates how, out of the millions of DNA regions, in which we differ from other apes, we can sift out those that, in response to a specific regulatory protein, cause gene activity changes in human cells. In this set, we find genes involved in brain and breast development and also in diseases like Alzheimer’s and Parkinson’s.” Reference

Deiodinase type 3 methylation increases in response to thyroid stimulating hormone in a fasting adapted mammal

Epigenetic regulation through the methylation of genomic cytosine nucleotides is a crucial component in the regulation of gene expression. DNA methylation in mammalian genomes serves as an important modulator of when and where a gene should be expressed, by primarily targeting 5′‐CpG‐3′ dinucleotides which have been associated with gene regulatory sites, including promoter sequences. Food deprivation in mammals is associated with increases in reverse triiodothyronine (rT3), an inactive form of the metabolism driving thyroid hormone triiodothyronine (T3), as a direct consequence of increases in the expression of the gene deiodinase type 3 (DI3). However, in prolong‐fasted, metabolically active elephant seal pups, cellular thyroid hormone‐mediated components are up‐regulated with fasting duration, while rT3 levels remain low. To address our hypothesis that methylation status of DI3 contributes to the silencing of a gene that would otherwise be expressed under such physiological conditions, we infused early fasted pups with thyroid stimulating hormone (TSH), extracted and treated genomic DNA with sodium bisulfite in an effort to detect and quantify 5‐methylcytosine (5mCs) at single base‐pair resolution. In reference to the genomic sequence of DI3 in elephant seals, prior to infusion with TSH, there is 50% methylation of 15/30 CpG sites in adipose tissue. Twenty‐four hours post infusion with TSH, the degree of methylation of DI3 increases to 93%, with methylation observed in 27/29 CpG sites. Moreover, in muscle, methylation reaches 90%, with 26/29 CpG sites being methylated at only 60 minutes post‐infusion with TSH, suggesting a tissue specific sensitivity. Aside from providing an initial and novel assessment of methylation regulation and status in elephant seals, the data demonstrates a unique adaptation to a methylation pattern where the compacted chromatin structure of DI3 plays a crucial role in suppressing the normal physiological response to fasting otherwise present in most mammals.Support or Funding InformationMinority Health and Health Disparities International Research Training Program, Dennis R. Washington Achievement Graduate Scholarship

First Genome-Wide Mutational Map of Macaques a Boon for Evolutionary Studies.

Scientists have long used comparative animal studies to better understand the nuances of human evolution, from making diverse body plans to the emergence of entirely powerful and unique features structures, including the human brain. Now, in a new study appearing in the advanced online edition of Molecular Biology and Evolution, corresponding authors Perdomo-Sabogal et al. (2016) have explored the global gene regulator GABPa to better understand its influence as a master switch. Their work honed in on human-specific DNA regions potentially contributing to human evolution in key functions including cell energy, division and death, and diseases ranging from brain disorders to diabetes. Many of the GABPa gene regulatory sites were found to be significantly enriched at genes important for brain, central nervous system and spinal cord functions that are unique to humans, an area ripe for future research, and GABPa deficits linked to Alzheimer’s and Parkinson diseases, breast cancer, and autism. GABPa exerts its profound genomic influence by a tandem repeat of four highly conserved DNA letters. The GGAA consensus motif is found to control genes in all primates, mice, dogs, and cows, underscoring its importance in these animals. By performing ChIP-Seq experiments in a human cell line, the group found that GABPa exercises its influence across the genome at 11,619 putative GABPa binding sites representing nearly 4,000 genes. Through a tour de force sequence comparison of the human GABPa binding sites with similar sequences from 34 mammals, they identified 224 GABPa binding sites unique to humans. Comparing chimpanzee, macaque, and human cell lines and mutating the GGAA sequence to more ancestral states to determine how these changes affected GABPa function, they further narrowed down their function. For instance, when the human GABPa binding sites were introduced into primate cells, gene expression was increased up to five times in 17 out of 18 cases, indicating that a recent human mutation in GABPa resulted in higher gene expression and forces behind the evolution of novel, uniquely human functions. “Mutations that cause changes in the regulation of gene activities are one of the major factors in shaping species during evolution,” said corresponding author Robert Querfurth. “Our study demonstrates how, out of the millions of DNA regions, in which we differ from other apes, we can sift out those that, in response to a specific regulatory protein, cause gene activity changes in human cells. In this set, we find genes involved in brain and breast development and also in diseases like Alzheimer’s and Parkinson’s.”

Enhancer Landscapes Reveal Transcription Factor Network Dependencies in Chronic Lymphocytic Leukemia

Genome sequencing efforts of chronic lymphocytic leukemia have revealed mutations that disrupt protein-coding elements of the genome (Puente et al, 2011; Wang et al, 2011; Landau et al, 2013). Recently, comprehensive whole-genome sequencing efforts have begun to reveal the genetic aberrations that occur outside of protein-coding exons, many that may perturb gene regulatory sites (Puente et al, 2015). These include enhancer elements that make physical contact with gene promoters to regulate gene expression in a cell-type specific manner. While mutations certainly promote CLL leukemogenesis, epigenomic alterations may also play an important role in facilitating disease progression and maintenance by inducing the gene expression aberrations that have long been observed in CLL. Epigenomic alterations include chromatin structure changes that facilitate altered transcription and chromatin factor recruitment to regulatory elements. While comprehensive genome-wide DNA methylation studies have been performed on human cancers and normal cell counterparts including CLL, other comprehensive studies of cancer epigenomes have been lacking. We have completed an analysis of chromatin structures in a cohort of primary chronic lymphocytic leukemia (CLL) samples with comparisons to normal CD19+ B lymphocytes (n = 18 CLL samples, n = 5 normal B lymphocyte samples). We used chromatin accessibility assays (ATAC-seq) and genome-wide enhancer mapping (H3K27ac ChIP-seq) to comprehensively define the transcriptionally active chromatin landscape of CLL. We have discovered greater than 15,000 novel regulatory elements when compared to previously annotated regulatory elements. Moreover, sites within the loci of several hundred genes were found to have large regions of gained chromatin accessibility and H3K27 acetylation, revealing the appearance of aberrant enhancer activity. These gained enhancer elements correspond with increased gene expression and are found at gene loci such as LEF1, PLCG1, CTLA4, and ITGB1. We have also systematically identified the super-enhancers of CLL - large complex regulatory regions that possess unique tissue-specific regulatory capabilities. Many of these super-enhancers are found in normal B lymphocytes, yet the super-enhancer at the ITGB1 and LEF1 loci are CLL-specific and may be considered to facilitate leukemia-specific expression. We have found CLL-specific enhancers are also significantly associated with annotated CLL risk variants, and have identified enhancer-associated SNPs found within CLL-risk loci predicted to disrupt transcription factor binding sites. These include SNPs at the IRF8 and LEF1 locithat lead to the creation and destruction of SMAD4 and RXRA binding sites, respectively. Additionally, we have analyzed whole-genome sequencing data from a subset of our sample cohort. Mutational hotspots in the CXCR4 and BACH2 promoters occur within open, acetylated regions. Moreover, we discover recurrent mutations in enhancers of the ETS1 and ST6GAL1 locus that have not been previously annotated. Using a transcription factor network modeling approach, we used these global chromatin structure characteristics to determine networks that are highly active in CLL. We find that transcription factors such as NFATc1, E2F5, and NR3C2 are among the most interconnected transcription factors of the CLL genome, and their connectivity is significantly higher in CLL cells compared to normal B cells. In contrast, network profiling of CLL cells predicts loss of MXI1 connectivity, a negative regulator of the MYC oncogene. By treating cells with specific pharmacological inhibitors of NFAT family members including cyclosporin and FK506, we are able to reduce NFAT-mediated network connectivity, resulting in a selective loss of NFAT-bound enhancers. This leads to CLL cell death in vitro of both cell lines and primary CLL patient samples. Our results reveal the unique chromatin structure landscape of CLL for the first time, and identify the CLL-specific enhancer elements that confer the transcriptional dysregulation that has long been observed in this disease. Use of these chromatin structure analyses and enhancer landscapes has allowed us to construct the intrinsic transcription factor network of CLL, and determine a particular dependency on NFAT signaling for cell survival. DisclosuresNo relevant conflicts of interest to declare.

Divergent androgen regulation of unfolded protein response pathways drives prostate cancer.

The unfolded protein response (UPR) is a homeostatic mechanism to maintain endoplasmic reticulum (ER) function. The UPR is activated by various physiological conditions as well as in disease states, such as cancer. As androgens regulate secretion and development of the normal prostate and drive prostate cancer (PCa) growth, they may affect UPR pathways. Here, we show that the canonical UPR pathways are directly and divergently regulated by androgens in PCa cells, through the androgen receptor (AR), which is critical for PCa survival. AR bound to gene regulatory sites and activated the IRE1α branch, but simultaneously inhibited PERK signaling. Inhibition of the IRE1α arm profoundly reduced PCa cell growth in vitro as well as tumor formation in preclinical models of PCa in vivo. Consistently, AR and UPR gene expression were correlated in human PCa, and spliced XBP-1 expression was significantly upregulated in cancer compared with normal prostate. These data establish a genetic switch orchestrated by AR that divergently regulates the UPR pathways and suggest that targeting IRE1α signaling may have therapeutic utility in PCa.

Epstein-Barr virus oncoprotein super-enhancers control B cell growth.

Super-enhancers are clusters of gene-regulatory sites bound by multiple transcription factors that govern cell transcription, development, phenotype, and oncogenesis. By examining Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), we identified four EBV oncoproteins and five EBV-activated NF-κB subunits co-occupying ∼1,800 enhancer sites. Of these, 187 had markedly higher and broader histone H3K27ac signals, characteristic of super-enhancers, and were designated "EBV super-enhancers." EBV super-enhancer-associated genes included the MYC and BCL2 oncogenes, which enable LCL proliferation and survival. EBV super-enhancers were enriched for B cell transcription factor motifs and had high co-occupancy of STAT5 and NFAT transcription factors (TFs). EBV super-enhancer-associated genes were more highly expressed than other LCL genes. Disrupting EBV super-enhancers by the bromodomain inhibitor JQ1 or conditionally inactivating an EBV oncoprotein or NF-κB decreased MYC or BCL2 expression and arrested LCL growth. These findings provide insight into mechanisms of EBV-induced lymphoproliferation and identify potential therapeutic interventions.

Inference of expanded Lrp-like feast/famine transcription factor targets in a non-model organism using protein structure-based prediction.

Widespread microbial genome sequencing presents an opportunity to understand the gene regulatory networks of non-model organisms. This requires knowledge of the binding sites for transcription factors whose DNA-binding properties are unknown or difficult to infer. We adapted a protein structure-based method to predict the specificities and putative regulons of homologous transcription factors across diverse species. As a proof-of-concept we predicted the specificities and transcriptional target genes of divergent archaeal feast/famine regulatory proteins, several of which are encoded in the genome of Halobacterium salinarum. This was validated by comparison to experimentally determined specificities for transcription factors in distantly related extremophiles, chromatin immunoprecipitation experiments, and cis-regulatory sequence conservation across eighteen related species of halobacteria. Through this analysis we were able to infer that Halobacterium salinarum employs a divergent local trans-regulatory strategy to regulate genes (carA and carB) involved in arginine and pyrimidine metabolism, whereas Escherichia coli employs an operon. The prediction of gene regulatory binding sites using structure-based methods is useful for the inference of gene regulatory relationships in new species that are otherwise difficult to infer.

Single-molecule analysis of transcription factor binding at transcription sites in live cells.

Although numerous live-cell measurements have shown that transcription factors bind chromatin transiently, no measurements of transient binding have been reported at the endogenous response elements (REs) where transcription is normally induced. Here we show that at endogenous REs, the transcriptionally productive specific binding of p53 and of the glucocorticoid receptor (GR) is transient. We also find residence times roughly comparable to that of endogenous GR REs at an artificial, multi-copy array of gene regulatory sites, supporting the use of multi-copy arrays for live-cell analysis of transcription. Finally, we find that at any moment only a small fraction of TF molecules are engaged in transcriptionally productive binding at endogenous REs. The small fraction of bound factors provides one explanation for gene bursting and it also indicates that REs may often be unoccupied, resulting in partial responses to transcriptional signals.

Do not put all teleosts in one net: Focus on the sox2 and pou2 genes

The Pou2 and Sox2 proteins are major transcription factors for development and cell differentiation. In teleosts, the expression patterns of pou2 or sox2 are different between species from distant families, suggesting different regulatory mechanisms of gene expression. In this study, we assessed the divergences among teleosts, including within closely related species. The pou2 and sox2 gene expression patterns were characterised over several developmental stages in a cyprinid model, i.e., the goldfish, and the potential regulation sites of these genes within teleost conserved regions were localised. During embryonic development, differences in the expression patterns between the goldfish and other teleosts, including zebrafish, were observed for both genes. The in silico analysis of the 5' flanking regions of the pou2 gene showed high conservation within teleosts, whereas the sox2 sequence diverged in tetraodontiforms. Certain putative cis regulatory elements were common to all teleosts, whereas others were found only in cyprinids. The analysis of the DNA methylation patterns of the pou2 and sox2 upstream sequences revealed that the studied CpG sites remained hypomethylated at all stages of embryo development in both genes. In contrast, in the adult fin, the studied CpG sites were hypermethylated in pou2 but not in sox2, suggesting the existence of methylation-sensitive regions in pou2. Overall, although most similarities at the level of the gene regulatory sites were found within cyprinids, the expression pattern of pou2 or sox2 during development differs between cyprinids species.

Genomics and Successful Aging: Grounds for Renewed Optimism?

Successful aging depends in part on delaying age-related disease onsets until later in life. Conditions including coronary artery disease, Alzheimer's disease, prostate cancer, and type 2 diabetes are moderately heritable. Genome-wide association studies have identified many risk associated single-nucleotide polymorphisms for these conditions, but much heritability remains unaccounted for. Nevertheless, a great deal is being learned. Here, we review age-related disease associated single-nucleotide polymorphisms and identify key underlying pathways including lipid handling, specific immune processes, early tissue development, and cell cycle control. Most age-related disease associated single-nucleotide polymorphisms do not affect coding regions of genes or protein makeup but instead influence regulation of gene expression. Recent evidence indicates that evolution of gene regulatory sites is fundamental to interspecies differences. Animal models relevant to human aging may therefore need to focus more on gene regulation rather than testing major disruptions to fundamental pathway genes. Recent larger scale human studies of in vivo genome-wide expression (notably from the InCHIANTI aging study) have identified changes in splicing, the "fine tuning" of protein sequences, as a potentially important factor in decline of cellular function with age. Studies of expression with muscle strength and cognition have shown striking concordance with certain mice models of muscle repair and beta-amyloid phagocytosis respectively. The emerging clearer picture of the genetic architecture of age-related diseases in humans is providing new insights into the underlying pathophysiological pathways involved. Translation of genomics into new approaches to prevention, tests and treatments to extend successful aging is therefore likely in the coming decades.