DOT1L is evicted during the 2-cell stage when cells are totipotent and massive epigenetic and transcriptional alterations occur. Embryonic stem cell lines that are derived from the blastocyst tolerate the loss of DOT1L, while the reduction of DOT1L protein levels or its catalytic activity greatly enhances somatic cell reprogramming to induced pluripotent stem cells. DOT1L knockout mice are embryonically lethal when organogenesis commences. We catalog the rapidly increasing studies of total and lineage specific knockout model systems that show that DOT1L is broadly required for differentiation. Reduced DOT1L activity is concomitant with increased developmental potential. Contrary to what would be expected of a modification that is associated with active transcription, loss of DOT1L activity results in more upregulated than downregulated genes. DOT1L also participates in various epigenetic networks that are both cell type and developmental stage specific. Taken together, the functions of DOT1L during development are pleiotropic and involve gene regulation at the locus specific and global levels.DOT1-Like (DOT1L) is the sole methyltransferase of histone H3K79, a modification enriched mainly on the bodies of actively transcribing genes. DOT1L has been extensively studied in leukemia were some of the most frequent onco-fusion proteins contain portions of DOT1L associated factors that mislocalize H3K79 methylation and drive oncogenesis. However, the role of DOT1L in non-transformed, developmental contexts is less clear. Here we assess the known functional roles of DOT1L both  Saccharomyces cerevisiae where its deletion redistributes repressive silent information regulator proteins from the telomere leading to activation of expression at those locations (Disruptor Of Telomeric silencing 1—Like (DOT1L) is an enigmatic histone methyltransferase that targets histone H3K79, a residue that is within the nucleosome core  and is cocations . DOT1L iocations . Unless ocations , it doesocations . Additioocations . Thus thocations ]. While ocations , methylaocations . Thus thin vivo, H3K79 methylation accumulates on the bodies of genes only after transcription commences  ActiCaenorhabditis elegans, H3K79me2 is enriched in both sperm and oocytes and is rapidly removed at 1-cell stage  stage of development. After the 2C stage, pericentromeric chromatin forms chromocenters of colocalized centromeres that appear as nuclear puncta. This reorganization is dependent on transcription of major satellite DNA from the paternal chromosome [reviewed: ]. DOT1L In vitro, 2C-like cells (2CLC) spontaneously occur at about a frequency of 0.5% from embryonic stem cells (ESCs) (in vitro (s (ESCs) . ESCs ars (ESCs) . Althougs (ESCs) . Moreoves (ESCs) . Genes ts (ESCs) . Pharmacs (ESCs) . DOT1L iin vitro . We notein vitro . Thus, tin vitro neuronal differentiation of ESCs  pathway, and aneuploidy suggesting disruption of chromosomal segregation . In this process, a small set of proteins - commonly the Yamanaka factors OCT4, SOX2, c-MYC, and KLF4 - are introduced into somatic cells to obtain iPSCs, which are functionally equivalent to ESCs, at a low efficiency (∼0.1%–5%). In 2012, gramming . Methylagramming , comparagramming . DOT1L igramming  or expregramming  demonstrDOT1L depletion and/or catalytic inhibition also enhances mouse reprogramming of tail tip fibroblasts , peripheThe second generation DOT1L inhibitor, SGC0946 (which is 10-fold more potent than EPZ004777) , was ideDot1l deletion and catalytic inactivation result in exceedingly few transcriptional changes in ESCs  Loss of H3K79me and gene expression of transcriptional repressor(s), 2) A consistent global reduction of transcription at all H3K79me-enriched genes such that genes without modification that do not change in expression appear elevated due to artifacts of bulk RNA-Seq , 3) A ne elegans , or 4) Aeq level . In factbination  indicatibination .Nfix, both a transcriptional activator and repressor   most potUnlike in reprogramming, DOT1Li treatment upregulated genes are already marked by H3K27me3 in ESCs , similarvia a complex containing the deacetylase SIRT1 and H3K9 methyltransferase SUV39H1  was completely lethal by E14.5-E15.5  mouse model of DOT1L histone methyltransferase activity (HMT) reveals specific effects on blood cell formation . Structuex vivo . Thus, D.5-E15.5 . A lower.5-E15.5 . TherefoDot1l reporter gene expression is uniform at the earliest timepoint assessed, E7.5, but is elevated in the extra-embryonic tissues of the visceral endoderm and mesoderm of the yolk sac, and in primitive erythrocytes by E9.5  H3K79me2/3 are rapidly lost post-fertilization in the absence of DNA replication. The current school of thought is that H3K79me is diluted by nucleosome turnover  but a poIn vitro, would increased DOT1L activity prevent cycling to the 2CLC?2) Reduced H3K79me is correlated with increased developmental potential , but is 3) H3K79me3 is found at pericentric heterochromatin in fibroblasts, oocytes , and ESC4) At the totipotent 2C stage, DOT1L is shuttled to the cytoplasm and enforced nuclear maintenance precipitates reorganization of pericentric heterochromatin to chromocenters. Does DOT1L have distinct functions throughout development and in different lineages?5) Does H3K79me2 function as a book-keeping epigenetic modification that denotes genes for expression rather than having a specific activating role? If true, nuclear DOT1L at the 2C stage could methylate the major satellite repeats before they are protected within the chromocenter, establishing an aberrant pattern of expression.6) H3K79me2 is enriched on the bodies of transcribed genes  and its via SIRT1/SUV39H1 (7) In leukemias, DOT1L activity was shown to be required for maintenance, but not activation, of gene expression . Is this/SUV39H1  in leuke8) DOT1L is only known to target H3K79 for methylation. Does DOT1L have additional histone or non-histone targets similar to other epigenetic modifiers?Increased developmental potential is correlated with reduced DOT1L activity . In the


Long regarded as “junk DNA,” transposable elements (TEs) have recently garnered much attention for their role in promoting genetic diversity and plasticity. While many processes involved in mammalian development require TE activity, deleterious TE insertions are a hallmark of several psychiatric disorders. Moreover, stressful events including exposure to gestational infection and trauma, are major risk factors for developing psychiatric illnesses. Here, we will provide evidence demonstrating the intersection of stressful events, atypical TE expression, and their epigenetic regulation, which may explain how neuropsychiatric phenotypes manifest. In this way, TEs may be the “bridge” between environmental perturbations and psychopathology. Transposable elements (TEs) are “selfish” sequences of DNA that replicate themselves across the genome. Barbara McClintock published the first account of TEs in 1950 after observing consistent breakages at certain loci in maize which had the ability to “transpose” and insert themselves into different positions along the genome. Interestingly, the loci also had a regulatory effect on the genes surrounding the new position; the expression of the adjacent genes was often lowered or even eliminated after these insertions. These were termed “mutable loci” or “controlling elements” are now classified as DNA transposons, which are one of the two major types of TEs. DNA transposons excise themselves from their position in the genome and mobilize to a different position via a “cut and paste” mechanism . The othRetrotransposons can be further classified as Long Interspersed Nuclear Elements (LINEs), Short Interspersed Nuclear Elements (SINEs), and Human Endogenous Retroviruses (HERVs). HERVs are the remnants of ancient exogenous retroviral infections and make up roughly 8% of the human genome . HERVs hSome families of HERVs however, remain transcriptionally active in the human genome and may be involved in cellular function and disease susceptibility  which wicis-regulatory elements, such as transcription factor binding sites, that serve as enhancers and promoters ultimately affecting the transcription of eukaryotic genes . This model involves injecting a viral mimetic into pregnant animals to elicit an immune response and is used to assess offspring outcomes following exposure to gestational infection . MIA is  of mice . AlthougSchizophrenia is a complex neurodevelopmental disorder that is characterized by the presence of symptoms like delusions, hallucinations, disorganized speech, and negative affect . While tMuch of the research surrounding TEs and schizophrenia illustrate a differential expression of HERVs. HERV-W in particular is elevated in the blood of subjects with recent-onset schizophrenia but not present in control individuals . There iRecently, PIWIL2 and MGARP in the DLPFC and anterior cingulate cortex of primates exposed to MIA  refers to a wide spectrum of neurodevelopmental disorders that are primarily characterized by impairments in communication and social interaction . SimilarHundreds of gene have been implicated in ASD etiology . One risMecp2 lead to the development of Rett syndrome  inhibitor valproic acid (VPA). ASD model mice demonstrated an upregulation in ERV expression in whole brain from gestational day 10 to adulthood  is a severe pathological condition that can develop after a major traumatic event . While ade novo in the hippocampus and in cell lines  .Additionally, administration of an IL-1 antagonist prevented SEFL . Other pWhether or not stress can mediate inflammation independently of LINE-1 however, remains to be elucidated. Nonetheless, this evidence suggests deleterious TE activity that intersects with region-specific glucocorticoid function and inflammation could be a risk factor for developing PTSD .Major depressive disorder (MDD) and bipolar disorder (BPD) are complex multigenic diseases that can arise from interactions between endogenous genetic risk factors, and exogenous risk factors such as childhood adversity and substance abuse . MDD hasDecreased expression of human endogenous retroviral protein HERV-W gag is present in brain of patients with major depression and BPD , althougin vitro  but not in healthy controls . It has Almost every psychiatric disease exhibits a sex difference in either its prevalence or symptomology. For example, boys are at a higher risk for disorders of development, such as autism, ADHD, or early onset schizophrenia, whereas women are more commonly diagnosed with stress related disorders like depression orPSTD . There iComprising almost half of the entire genome, TEs have garnered substantial interest given their involvement in psychiatric disease. Mounting evidence demonstrates interactions with the stress system, in both early development and adulthood, leads to deleterious TE activity that coincide with permanent changes in brain physiology, especially inflammation, leading to pathology. Inflammation may also be induced by the activation of cellular innate immunity by ectopic expression of TE RNAs in a number of tissues. Further, TEs contribute to somatic mosaicism, making them an appealing avenue to explore individual differences in both psychiatric diseases and in normal variation of cognitive and behavioral traits. However, substantial research needs to be done, as TEs have received a miniscule fraction of the research attention that protein coding elements of the genome have to date. If, as their evolutionary origins would suggest, TEs are acutely sensitive to host stress, then they represent a means by which the environment can program the genome across the lifespan, for both good and ill, a hypothesis that deserves a much deeper analysis.


LIPA, is the sole neutral lipid hydrolase in lysosomes, responsible for cleavage of cholesteryl esters and triglycerides into their component parts. Inherited forms of complete  or partial LAL deficiency  are fortunately rare. Recently, LAL has been identified as a cardiovascular risk gene in genome-wide association studies, though the directionality of risk conferred remains controversial. It has also been proposed that the low expression and activity of LAL in arterial smooth muscle cells (SMCs) that occurs inherently in nature is a likely determinant of the propensity of SMCs to form the majority of foam cells in atherosclerotic plaque. LAL also likely plays a potential role in fatty liver disease. This review highlights the nature of LAL gene mutations in WD and CESD, the association of LAL with prediction of cardiovascular risk from genome-wide association studies, the importance of relative LAL deficiency in SMC foam cells, and the need to further interrogate the pathophysiological impact and cell type-specific role of enhancing LAL activity as a novel treatment strategy to reduce the development and induce the regression of ischemic cardiovascular disease and fatty liver.Lysosomal acid lipase (LAL), encoded by the gene  LIPA, is the sole lysosomal lipase, responsible for the critical functions of hydrolysis of cholesteryl esters (CE) contained in endocytosed lipoproteins to component free cholesterol and fatty acids, and of triglycerides to their component fatty acids. LAL also cleaves stored CE delivered to lysosomes for breakdown in the lipophagy component of autophagy , encoded by the gene utophagy , enhanceutophagy . Completutophagy . Near toutophagy . RecombiLIPA gene is located on chromosome 10q23.2–23.3  and ABCG1 that promote cholesterol efflux from cells . LAL theom cells . LAL is om cells . Due to om cells , LAL is om cells . Injecti in vivo .LAL is similarly necessary for metabolism of lipids contained in apoptotic cells endocytosed by macrophages during efferocytosis . In lipoLIPA DNA methylation in infants  and Cholesteryl Ester Storage Disease (CESD). They are referred to collectively as LAL deficiency (LALD). WD involves complete loss or less than 1% preservation of LAL activity  causing feedback inhibition of HMG-CoA reductase which reduces cholesterol synthesis, LDL receptor downregulation to reduce LDL uptake, acyl-cholesterol acyltransferase activation to esterify free cholesterol, and downregulation of fatty acid synthesis . In LALDLIPA gene, and can be distinguished biochemically by the level of residual LAL activity. CESD is therefore not the heterozygous form of WD, but is less severe due to the nature of the LIPA mutations present in both alleles. WD mutations are more often frame-shift, nonsense, or splice-site mutations rather than missense mutations, which are more likely to result in CESD  carriers ; the fre 160,000 . It is e 160,000 . There a 160,000 , and the 177,452 , while a 177,452 . It is cSeveral recent studies screening patients with hypercholesterolemia or elevated transaminases using the dried blood spot assay (DBS) for blood LAL activity  have faiLIPA gene sequencing reveals a pathogenic variant. The reader may refer to diagnostic flowcharts published by Reiner et al. and Lipiński et al.  rs1412444 and rs2246833 on chromosome 10q23 as common variants associated with coronary artery disease (CAD) risk  of large European, South Asian and Mexican cohorts have identified AD) risk . While tonocytes . No clealatation . In contlatation , CAD risIn vitro studies to determine the effect of LIPA risk variants on LAL function and activity and cell cholesterol metabolism have reached differing conclusions. Morris et al. investigated a coding variant rs1051338, which is in high linkage disequilibrium (r2 = 0.89) with the GWAS variant rs2246833 and causes a nonsynonymous threonine to proline change within the signal peptide of LAL , even lower in NASH patients (∼20%), and further reduced in patients with cirrhosis (∼50%)  found in the general population is now being investigated. NAFLD is characterized by fat accumulation or steatosis in the liver, and has a worldwide prevalence of approximately 25% . The begs (∼50%) . Lower Ls (∼50%) . Some sts (∼50%)  and knows (∼50%) . Gravito markers . LAL is  markers . ContinuIt should be noted that there are limitations to the DBS assay for LAL activity. DBS assays testing for other lysosomal storage diseases mostly reflect leukocyte enzyme activity, and since activity is normalized not to protein content or cell counts but to blood spot area, blood leukocyte count could importantly influence results . PlateleLIPA variants in the NHGRI-EBI GWAS catalog (et al. suggests a post-translational mechanism, demonstrated using in vitro and mouse models, and confirmed in NAFLD patient biopsies, that NAFLD is associated with low functional levels of LAL protein and accumulation of dysfunctional ubiquitinated LAL (et al. report that low LAL activity in liver biopsies of NAFLD patients was shown to be independent of LAL expression (et al. show an association of elevated LDL with low LAL activity in NAFLD patients (et al. similarly associate low LAL with high BMI in NAFLD patients, and LIPA variants have previously been associated with metabolic syndrome, though this association requires further investigation (et al. also demonstrate increased necroinflammation in NASH patients with lower LAL activity, which may speak to the immune roles of LAL (The pathogenesis of the lowered LAL activity in NAFLD is as yet unknown. Multiple studies screening for LAL mutations in NAFLD patients with low LAL activity did not find any , and the catalog , so it dated LAL . In agrepression . Other epression . This supression . We havepatients . Thoen etigation . Thoen es of LAL . It is us of LAL .et al. speculate that determining the epigenetic or metabolic factors leading to low LAL may be instrumental in treating NAFLD, and suggest that sebelipase alfa enzyme-replacement therapy may be useful in these patients (In vitro, a novel GATA3-binding molecule morroniside increases LIPA expression and reduces the fibrosis response in hepatic stellate cells (Baratta patients . Sebelippatients ; howeverpatients . Both stte cells . FurtherLIPA expression as a predictor of cardiovascular risk, and the likelihood that naturally occurring low levels of LIPA expression predict the formation of SMC foam cells, the proposed primary source of foam cells in atherosclerosis. It is likely that in the next decade the activity and functions of LAL will assume an ever increasing role in our understanding of the pathogenesis and potential treatment of both atherosclerosis and fatty liver disease.LAL is critical to life as the sole neutral lipid hydrolase in lysosomes, as a director of downstream cholesterol metabolism in cells, and as a driver of total body reverse cholesterol transport. Total deficiency of LAL is fatal, whereas even very low levels of residual LAL activity are compatible with life though predisposing to liver disease and potentially atherosclerosis. In addition to utilizing recombinant LAL to treat complete and partial LAL deficiency, we are now learning much more about the role of


Substance abuse and addiction represent a significant public health problem that impacts multiple dimensions of society, including healthcare, the economy, and the workforce. In 2021, over 100,000 drug overdose deaths were reported in the US, with an alarming increase in fatalities related to opioids and psychostimulants. Understanding the fundamental gene regulatory mechanisms underlying addiction and related behaviors could facilitate more effective treatments. To explore how repeated drug exposure alters gene regulatory networks in the brain, we combined capped small (cs)RNA-seq, which accurately captures nascent-like initiating transcripts from total RNA, with Hi-C and single nuclei (sn)ATAC-seq. We profiled initiating transcripts in two addiction-related brain regions, the prefrontal cortex (PFC) and the nucleus accumbens (NAc), from rats that were never exposed to drugs or were subjected to prolonged abstinence after oxycodone or cocaine intravenous self-administration (IVSA). Interrogating over 100,000 active transcription start regions (TSRs) revealed that most TSRs had hallmarks of bonafide enhancers and highlighted the KLF/SP1, RFX, and AP1 transcription factors families as central to establishing brain-specific gene regulatory programs. Analysis of rats with addiction-like behaviors versus controls identified addiction-associated repression of transcription at regulatory enhancers recognized by nuclear receptor subfamily 3 group C (NR3C) factors, including glucocorticoid receptors. Cell-type deconvolution analysis using snATAC-seq uncovered a potential role of glial cells in driving the gene regulatory programs associated with addiction-related phenotypes. These findings highlight the power of advanced transcriptomics methods to provide insight into how addiction perturbs gene regulatory programs in the brain. Drug addiction and related health problems impact millions of lives in the United States and impose an enormous medical, social, and economic burden on society . AddictiNumerous studies have demonstrated that long-lasting changes in gene expression patterns in brain regions of the reward pathway are a critical mechanism by which substances of abuse lead to persistent drug-induced neuroadaptations . These nDespite this knowledge, remarkably little is known about the gene regulatory mechanisms responsible for driving these changes. Mammalian gene expression programs are orchestrated by the collective action of tens or even hundreds of thousands of regulatory elements, most of which are annotated as putative enhancers and located in regions far from the promoter regions of genes . EnhanceTo improve our understanding of gene regulation underlying addiction-related behaviors, we profiled the activity of regulatory elements in the brains of rats exhibiting addiction-like behaviors using a recently developed technique called capped small(cs)RNA-seq . csRNA-sHere, we compared transcription initiation profiles by csRNA-seq using brain tissues isolated from rats that were not exposed to drugs or were subjected to a well-validated extended access model of intravenous self-administration (IVSA) of oxycodone or cocaine . TissuesTo probe if substance abuse can alter gene regulatory programs in the brain, we comprehensively profiled active regulatory elements in two brain regions implicated in addiction: the prefrontal cortex (PFC) and nucleus accumbens NAc, . SamplesNr4a1 locus  which we refer to as Transcriptional Start Regions (TSRs). While 15.7% TSRs  in PFC and 19.5% TSRs  in NAc were within or proximate to annotated gene promoter regions, the majority were at promoter-distal sites within introns and intergenic regions of the genome  for transcribed accessible regions (n = 91323 ATAC-seq peaks that overlapped a TSR) compared to those that were not transcribed  genome organization can be an essential factor in gene regulation . To placNeurod6 gene locus were highly transcribed in the PFC but not in the NAc, while the dopamine receptor-1 (Drd1) gene locus was highly transcribed in the NAc but not in the PFC . To further support GR’s potential role in regulating these TSRs, several downregulated TSRs were found in the intergenic region upstream of Fkbp5  showed accessible chromatin exclusively in the expected cell types that directly overlapped TSRs identified in the bulk csRNA-seq experiments  with genome structure (HiC) and single-cell epigenomic data (snATAC-seq), the analysis of the regulatory landscape not only provided a comprehensive catalog of eRNAs but also identified TFs that are likely to play important regulatory roles. Using this approach, we discovered that GR-bound enhancers are strongly down-regulated during prolonged abstinence from oxycodone or cocaine IVSA, and that many of the impacted sites are specific to glial cells.There is firm evidence supporting the role of cell type- or stimulus-specific enhancers in the gene regulation , but detThe major finding of this study is the identification of TF-regulatory networks associated with a history of addiction-like behavior. The analysis of drug-altered TSRs revealed that GR-regulated enhancers were consistently repressed in PFC and NAc from rats with a history of oxycodone and cocaine addiction-like behavior compared to controls. This result is consistent with converging evidence that the brain stress system involving glucocorticoid signaling plays a critical role in the development of addiction in humans and rodent models of addiction-like phenotypes . The celn = 2/condition), which may lead to a low statistical power to detect differentially expressed TSRs and could explain why, despite identifying over 100,000 TSRs across two brain regions, we only detected a relatively small number of differentially regulated TSRs in both PFC and NAc. A study with a larger cohort of rats would be ideal for confirmation. Second, the control animals used in this study are rats that were never exposed to drugs; thus, our study design does not consider environmental factors associated with the behavioral protocol . Including rats with a low addiction index subjected to the same behavioral protocol but do not develop addiction-like phenotypes would serve as important control to provide more substantial evidence that the differences we observe reflect molecular changes associated with addiction-related processes rather than other phenomena. Third, different subregions of the PFC    and NAc orbital)  are knoworbital) .In summary, we used an unbiased and highly sensitive method to identify active enhancers by measuring levels of initiating transcripts from brain tissues of rats with addiction-like phenotypes. We identified TF-centered regulatory mechanisms implicated in addiction, including those regulated by GR in glial cells. Overall, our study demonstrates that transcriptional initiation profiling has the potential to dissect the gene regulatory mechanisms driving addiction-related phenotypes in an unbiased and quantitative manner.1,2 tissue repositories at UCSD and are part of an extensive and ongoing study of addiction that uses outbred HS rats3 (°C). Cryosections of approximately 500 microns were used to dissect PFC and NAc punches on a −20°C frozen stage. Bregma for PFC: 4.20-2.76 mm, and for NAc: 2.28-0.72 mm (3 sections were combined for each).Brain samples from male heterogeneous stock (HS) rats  were obtained from the cocaine oxycodoneHS rats3 . We seleHS rats3 . In thisHS rats3 . HS ratsWe extracted total RNA from PFC and NAc tissues dissected from 6 rat brains using Trizol Reagent  and Zirconium Beads RNase Free  with the Bullet Blender Blue  at speed 6 for 1 min. The RNA was purified according to the manufacturer’s instructions (Invitrogen).csRNA-seq was performed as described previously . Briefly® Stranded mRNA Prep . Samples were processed following the manufacturer’s instructions. The resulting libraries were multiplexed and sequenced with 100 basepairs (bp) Paired-End reads (PE100) to a depth of approximately 25 million reads per sample on an Illumina NovaSeq 6000. Samples were demultiplexed using bcl2fastq Conversion Software .RNA sequencing libraries were generated using the Illumina°C. PFC punches were dissected on a −20°C frozen stage. Tissues were then pulverized in liquid nitrogen. The Arima-Hi-C kit was used to construct the Hi-C libraries . Sequencing of the libraries was conducted on an Illumina Novaseq S4 instrument by Novogen Inc. The use of rodents was approved by UTHSC IACUC.One adult SHR/OlaIpcv naive rat was used to generate the Hi-C data. This rat was bred at the University of Tennessee Health Science Center using breeders provided by the Hybrid Rat Diversity Program at the Medical College of Wisconsin. The animal was fully anesthetized by using isoflurane before the brains were removed. Brain tissue was extracted and rapidly frozen. Cryosections of approximately 120 microns were obtained in a cryostat set at −11PFC brain tissue from one naive male HS rat was used to generate a single-nuclei ATAC-seq library. Nuclei were isolated from brain tissue as previously described . BrieflySequencing reads were trimmed for 3′ adapter sequences using HOMER (“homerTools trim −3 AGATCGGAAGAGCACACGTCT -mis 2 –min Match Length 4 -min 20”) and aligned to the rat mRatBN7.2/rn7 genome assembly using STAR  with defThe resulting normalized data was used for all downstream analyses. Normalized genome browser visualization tracks were generated using HOMER’s makeMultiWigHub.pl tool . TSR genTo identify differential TSRs between brain regions or conditions (naive vs. cocaine or oxycodone), we used DESeq2 with FDR < 10% PFC vs. NAc, Naive vs. Oxycodone, Naive vs. Cocaine, or Oxycodone vs. Cocaine, and 2-fold change, as cutoffs. DESeq2 log2 fold change, p-value, and adj. P-value for all differentially regulated TSRs in response to addiction-like behaviors in each tissue are reported in For RNA-seq analysis, sequencing reads were aligned to the rat mRatBN7.2/rn7 genome assembly using STAR  with defRaw FASTQ files associated with public ChIP-seq and ATAC-seq datasets were downloaded from NCBI’s Short Read Archive and processed in a consistent manner to ensure differences in data processing were minimized for downstream analysis. Reads from ChIP-seq or ATAC-seq datasets were analyzed in a consistent manner. Reads were first trimmed for adapter sequences and then aligned to the rat genome using STAR  with defDatasets used in the study include GR ChIP-seq GSE160806 from the rat hippocampus . ATAC-seHi-C reads were first trimmed for sequences downstream of the restriction/ligation site (“GATCGATC”) and aligned to the rat genome using STAR with default parameters. Normalized interaction contact maps were then generated using HOMER. PCA compartment analysis and topological domain (TAD) calls were generated using HOMER’s runPCAhic.pl and findTADsAndLoops.pl scripts . The sigde novo motif discovery of TSRs were performed using HOMER’s findMotifsGenome.pl tool using 200 bp sequences centered on  relative to TSR primary initiation sites  or from −100,100 relative to the center of ATAC-seq peaks .To analyze motif enrichment associated with changes in transcription levels, we analyzed regulated TSRs with MEIRLOP . SequencFurthermore, we provide the BigWig track with the map of transcription factor binding site predictions in the rat genome (rn7), which can be uploaded as a custom track on the UCSC browser as follow:track type=bigBed name=“HOMER Known Motifs rn7 (210922)” description=“HOMER Known Motifs rn7 (210922)”http://homer.ucsd.edu/homer/data/motifs/homer.KnownMotifs.rn7.210922.bigBed visibility=3bigDataUrl=Rattus norvegicus, built from the Ensembl Rnor 6.0 release 103 genome and annotation. The filtered results were subsequently analyzed using Signac 1.4.0 , Flt1 for endothelial cells. Pseudo bulk peak positions for each cell type were identified using MACS2  can be found below: The animal study was reviewed and approved by the institutional Animal Care and Use Committee at the University of California, San Diego.FT conceived, designed, and coordinated the study. CB conceived, designed, conducted the overall bioinformatic analysis. SD generated the csRNA-seq data. PM-P assisted with the csRNA-seq data generation. MC conducted the snATAC-seq analysis. HL generated snATAC-seq data. HC generated the HiC data. LC dissected the brain tissues. GG, OG, and AP contributed expertise important for the study design. FT, CB, and SD wrote the manuscript with input from all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A mutation in the chromatin remodeler chromodomain helicase DNA-binding 7 (CHD7) gene causes the multiple congenital anomaly CHARGE syndrome. The craniofacial anomalies observed in CHARGE syndrome are caused by dysfunctions of neural crest cells (NCCs), which originate from the neural tube. However, the mechanism by which CHD7 regulates the function of human NCCs (hNCCs) remains unclear. We aimed to characterize the cis-regulatory elements governed by CHD7 in hNCCs by analyzing genome-wide ChIP-Seq data and identifying hNCC-specific CHD7-binding profiles. We compared CHD7-binding regions among cell types, including human induced pluripotent stem cells and human neuroepithelial cells, to determine the comprehensive properties of CHD7-binding in hNCCs. Importantly, analysis of the hNCC-specific CHD7-bound region revealed transcription factor AP-2α as a potential co-factor facilitating the cell type-specific transcriptional program in hNCCs. CHD7 was strongly associated with active enhancer regions, permitting the expression of hNCC-specific genes to sustain the function of hNCCs. Our findings reveal the regulatory mechanisms of CHD7 in hNCCs, thus providing additional information regarding the transcriptional programs in hNCCs. Enhancers are genetic elements that were historically defined as DNA sequences that potentiate the cell type-specific transcription of transcriptional start sites (TSSs) located distally to the enhancer regions2. Although enhancers are responsible for exerting cell type-specific gene expression, they need to be accessible by adapting the local chromatin structure and enabling a precise response to external stimuli during development4. Accordingly, chromatin-based epigenetic alterations are closely linked to enhancer activity. The importance of chromatin regulation is also evidenced by the fact that defects in chromatin regulators cause genetic developmental disorders called chromatinopathies6. Notably, a genetic mutation in chromatin remodelers was observed in a subset of chromatinopathies, including ATRX syndrome, Floating-Harbor syndrome, and CHARGE syndrome5.Cell type-specific transcriptional responses regulate cellular identity, which is dynamically coordinated during the developmental processCHD78. CHD7 is a member of the chromodomain helicase DNA-binding (CHD) family, a group of ATP-dependent chromatin-remodeling factors that alter chromatin structure by rearranging the position and organization of nucleosomes on DNA9.CHARGE syndrome is a congenital disorder caused by a heterozygous mutation in 10. Notably, CHD7 binds and regulates the activity of central nervous system-specific enhancers to control the differentiation capacity of hNECs10, indicating an essential role of CHD7 in regulating enhancers, at least in the central nervous system.We previously demonstrated an epigenetic landscape regulated by CHD7 in human induced pluripotent stem cell (hiPSC)-derived neuroepithelial cells (hNECs)8, and the role of CHD7 in regulating the epigenetic signature of NCCs has thus been of interest. The pioneering work by Wysocka’s team using an in vitro differentiation system from human embryonic stem cells demonstrated that the formation of NCCs required the function of CHD711. Additionally, we previously demonstrated defective migration of human NCCs (hNCCs) derived from iPSCs from patients with CHARGE syndrome12. These results provide potential explanations for the pathology of CHARGE syndrome. However, although CHD7 has been shown to be essential for the function and development of hNCCs, the comprehensive genomic map governed by CHD7 in hNCCs is still unclear.Dysfunction of neural crest cells (NCCs) is a primary cause of CHARGE syndrome10. However, detailed information on the cell type-specific role of CHD7, especially in hNCCs, was not determined. In the current study, we aimed to compare CHD7-binding profiles in hiPSCs, hNECs, and hNCCs to identify hNCC-specific targets of CHD7. These findings highlight the fundamental role of CHD7 in cell type-specific gene regulation in hNCCs.We previously optimized the experimental conditions of chromatin immunoprecipitation (ChIP) followed by massive parallel DNA sequencing (ChIP-seq) for CHD7 and successfully generated CHD7-binding profiles in hNECs18, and identifying the cell-specific targets of CHD7 is thus necessary to elucidate its function. We therefore investigated the function of CHD7 in hNCCs using the ChIP-seq profiles of CHD7 in hiPSCs, hNECs, and hNCCs10 with transcriptome information using published datasets  of 31,518 peaks in hiPSCs, 11,529 (64.3%) of 17,941 peaks in hNECs, and 3,953 (73.8%) of 5,357 peaks in hNCCs) 26 in hNCCs, respectively  specification27. As noted above, dysfunction of hNCCs has been reported to be involved in the pathogenesis of CHARGE syndrome29. A potential link between CHD7 and TFAP2A might thus provide potential mechanistic insights into the pathogenesis of CHARGE-derived hNCCs.To gain further insights into the functional properties of CHD7, we cross-referenced CHD7-bound regions with the genome-wide chromatin state of histone modification in hiPSCs, hNECs, and hNCCs Fig. A. We genCs) Fig. . A totalGene Ontology (GO) analysis for hiPSCs and the hNEC-specific CHD7-regulated region revealed a similar trend, indicating that the sites were associated with neural differentiation, including “neurogenesis,” “nervous system development,” and “neuron differentiation” Fig. D. In con31. Moreover, we previously identified CHD7 as one of the components enriched in SEs across the hNEC genome10. This prompted us to determine if CHD7 was also enriched in SEs in hNCCs. We delineated SEs in hNCCs based on H3K27ac abundance using the ROSE algorithm, and identified 1,394 SEs  conferred cellular identities by regulating tissue-specific gene expression234 Fig. . FurtherWe further elucidated the role of CHD7 in transcriptional regulation by examining the expression of cell type-specific CHD7 target genes. First, we determined cell type-specific CHD7 target genes based on the ChIP-seq information for hiPSCs, hNECs, and hNCCs  (238 upregulated and 100 downregulated) in CHARGE-iPSC-NCCs12. We therefore considered that it would be interesting to determine if the datasets obtained in the current study could help to elucidate the molecular mechanism underlying the cellular phenotypes of CHARGE-iPSC-NCCs. We performed analyses to integrate these data and found that, although CHD7-bound genes were expected to be downregulated in CHARGE syndrome, the downregulated DEGs were not included in the CHD7-bound gene lists, while about ten upregulated DEGs in CHARGE-iPSC-NCCs were included as CHD7-bound genes (data not shown).We previously demonstrated the defective migration of iPSC-NCCs derived from CHARGE patients (CHARGE-iPSC-NCCs)Furthermore, we also examined the expression of genes bound by CHD7, TFAP2A, and NR2F1/2. Although a subset of genes was slightly downregulated in CHARGE-iPSC-NCCs, there was no major difference in the expression of these target genes  and plated on iMatrix511 (Nippi)-coated dishes in StemFit (AK03N) medium at feeder-free condition. The hiPSC-derived NECs were established and maintained as described previously54. The hNECs were passaged every 4 days and plated at a ratio of 1:5. The cells were dissociated using TrypLE Select (Thermo Fisher) and plated on Matrigel (Corning)-coated dishes in RHB-A medium (Takara) supplemented with 10 ng/mL EGF (Peprotech) and 10 ng/mL FGF2 (Peprotech). The hiPSC-derived NCCs were established previously and passaged less than three times for analysis. The hNCC medium consisted of 1:1 neurobasal medium (Thermo Fisher Scientific) and D-MEM/Ham’s F-12 (Wako) medium containing 1× GlutaMax (Thermo Fisher Scientific), 0.5× GEM 21 NeuroPlex serum-free supplement (Gemini Bio Products), 0.5× N2 supplement (Thermo Fisher Scientific), 5 mg/mL insulin (Sigma-Aldrich), 0.5% penicillin and streptomycin , 20 ng/mL FGF2 and 20 ng/mL EGF. The hNCCs were dissociated using TrypLE Select and plated on fibronectin (Sigma-Aldrich)-coated dishes.The hiPSC line 1210B2 was maintained as described previouslySelected genomic regions near the human MSX2 and GPC1 genes were amplified by PCR from the human genomic DNA and cloned into a pGL3 promoter vector (Promega). Primers used for cloning were listed in Supplemental Table hiPSCs, hNECs, and hNCCs were replated onto 12-well plates (Greiner Bio-One) and transfected with a reporter vector the next day. The sea pansy luciferase gene conjugated with human elongation factor-1α promoter (R-Luc) was also cotransfected as an internal control. Transfection was performed using a genejuice (Merck) according to the manufacturer’s procedures. Cells were solubilized 24 h after the transfection, and luciferase activity was measured according to the procedures recommended for the Dual-Luciferase Reporter Assay System (Promega) using GLOMAX multi-detection system (Promega). Firefly luciferase activity was divided by R-Luc activity from the cotransfected control plasmid.39. The dual cross-linking method was performed using 200 mM EGS for 30 min followed by 1% formaldehyde for 10 min and quenched by adding glycine to a final concentration of 125 mM. Cells were collected with NP-40 buffer  and centrifuged to a pellet, lysed in SDS lysis buffer , and then incubated for 10 min on ice. Cell lysates were sonicated using Covaris S2 (Covaris) until the DNA fragments were 200—300 base pairs in length. These chromatin samples were diluted 1:10 with dilution buffer , and 5% of the total volume was stored as input. The first immunoprecipitation was performed at 4 °C overnight with 3 μγ of an antibody conjugated with Dynabeads (VERITAS). Immune complexes were washed sequentially RIPA low-salt , RIPA high-salt , TE and eluted by TE/10 mM DTT at 37 °C for 30 min. Eluted chromatin was diluted 20 times with RIPA row-salt buffer for the second immunoprecipitation reaction. Next, immune complexes were washed, and immunoprecipitated DNA was eluted with elution buffer  at 65 °C for 4 h. DNA was further incubated with RNase and proteinase K  and purified using a ChIP DNA clean and concentrator kit (Zymo Research). Antibodies used in this study have been reported as suitable for ChIP assay: anti-CHD7 , anti-TFAP2A , anti-NR2F1  and anti-NR2F2 . Quantitative PCR (qPCR) analysis was performed using a ViiA7 system (Applied Biosystems) with KAPA SYBR FAST Master Mix (KAPA Biosystems). The primers used are listed in Supplemental Table Chromatin immunoprecipitation was performed as previously described with minor modifications55. Cell type-specific CHD7-peaks were identified using the DiffBind (v. 3.6.1) R package. Overlaps for each peak were calculated using bedtools (v. 2.17.0) with default parameters. The genome-wide peak distribution from TSS was calculated using the ChIPseeker (v. 1.32.0) R package, and functional GO analysis of CHD7 peaks was performed using the GREAT website (http://bejerano.stanford.edu/great/public/html/index.php). The enhancer category was calculated based on the overlap of the CHD7-binding site and the enhancer region registered in the VISTA enhancer browser.ChIP-seq data for histone modification in hNCCs were obtained from the Gene Expression Omnibus (GEO) database (GSE28876). Other ChIP-seq data for CHD7-binding in hNCCs and other ChIP-seq data for hiPSCs and hNECs were deposited as a part of our previous study through GEO accession number GSE108506. Raw reads were trimmed based on read length and quality using Trimmomatic (v. 0.33). The trimmed reads were then aligned to the reference genome (UCSC hg19) using Bowtie2 (v. 2.1.0) with the default parameters, and only uniquely mapped reads were used for subsequent analyses. The resulting SMA files were converted to the BAM format using SAMtools (v. 0.1.19). Peak calling was performed using MACS2 (v. 2.2.7.1) with default settingsHeatmaps for each sample against the CHD7-binding region were made using deepTools (v. 1.5.11). The average H3K27ac read density at each region and the corresponding flanking region were calculated (bin size = 50). The lengths of the SE regions (between TSS and TES) were scaled relative to the median length.Enriched sequences in the top 500 CHD7-binding regions were searched using MEME (v. 1.4) software and the identified sequences were compared using motif databases (JASPAR and HOCOMOCO) with TOMTOM (v. 2.2) software.31. We used a promoter-exclusion zone of 5000 bp to prevent any enhancer contained within a window of ± 2500 bp around an annotated TSS from being stitched. Enhancer-associated genes were defined based on the calculated distance from the center of the SE to the nearest TSS of each gene. The assignment of enhancers to the closest genes in hNCCs is shown in Supplementary Table SEs were identified using the ROSE algorithm with default parameters based on H3K27ac intensity, in which enhancer peaks located within 12.5 kb were stitched together and ranked based on their input-subtracted signal for H3K27acSamples for RNA-seq were prepared using the TruSeq RNA Library Prep Kit (Illumina) according to the manufacturer’s protocol. The sequencing library was sequenced on a NestSeq 1000 (Illumina). The adapter sequence and low-quality reads were removed using trimmomatic (v. 0.33), and the trimmed reads were mapped to reference genome hg38 using Kallisto (v. 0.46.2) with default settings. The count matrix data from Kallisto was loaded into R software (v. 4.2.0), and downstream analysis was performed. Differentially expressed genes were evaluated using the limma (v. 3.52.2) library.Our previously published gene expression profiles of WT and CHARGE patient-derived NCCs data (GSE86212) were loaded into R (v. 4.2.0) for statistical analysis. Boxplots were made using the R package ggplot2 (v. 3.3.6).Supplementary Legends.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Figures.


SoxC-knockout FLS was used to identify the SOX4-dependent and independent aspects of the TNF-regulated transcriptome. We found that SOX4 and RELA physically interact with each other on the chromatin. Interestingly, ChIP-seq assays revealed that 70.4% of SOX4 peak summits were within 50bp of the RELA peak summits suggesting that both proteins bind in close-proximity on regulatory sequences, enabling them to co-operatively regulate gene expression. By integrating the ChIP-seq results with RNA-seq from SoxC-knockout FLS we identified a set of TNF-responsive genes that are targets of the RELA-SOX4 transcriptional complex. These TNF-responsive and RELA-SOX4-depenedent genes included inflammation mediators, histone remodeling enzymes and components of the AP-1 signaling pathway. We also identified an autoregulatory mode of SoxC gene expression that involves a TNF-mediated switch from RELA binding to SOX4 binding in the 3’ UTR of Sox4 and Sox11 genes. In conclusion, our results show that SOX4 and RELA together orchestrate a multimodal regulation of gene expression downstream of TNF signaling. Their interdependent activities play a pivotal role in the transformation of FLS in arthritis and in the inflammatory pathology of diverse tissues where RELA and SOX4 are co-expressed.SOX4 belongs to the group C of the SOX transcription factor family. It is a critical mediator of tumor necrosis factor alpha (TNF)-induced transformation of fibroblast-like s-ynoviocytes (FLS) in arthritis. In this study we investigated the genome wide association between the DNA binding and transcriptional activities of SOX4 and the NF-kappaB signaling transcription factor RELA/p65 downstream of TNF signaling. We used ChIP-seq assays in mouse FLS to compare the global DNA binding profiles of SOX4 and RELA. RNA-seq of TNF-induced wildtype and  SoxC family  of genes in the FLS inhibits synovitis and articular cartilage degeneration in TNF-induced arthritis in mice (Fibroblast-like synoviocytes (FLS) are cells that reside in the synovial lining of joints. During homeostasis, FLS maintain the composition of synovial fluid, produce joint lubricating and cartilage protecting proteins. However, during arthritic diseases they undergo epigenetic changes and transform into pathological cells . The tra in mice . The goaSox4, Sox11 and Sox12 are co-expressed in various types of progenitor cells, and act largely in redundancy to determine the behavior and survival of the progenitor cells  mice according to a well-established protocol  at Emory University. FLS were prepared either from wild type or protocol , 22. Brifl/fl FLS at passage 3 were infected with AdCMV5eGFP (control) or AdCMV5Cre (SoxC-knockout) adenovirus  at a concentration of 200 pfu/cell, for a period of 24h. FLS were treated with or without 10ng/mL recombinant human TNF (R&D Systems) for the last 16h. Total RNA was extracted and purified using Direct-zol RNA MicroPrep (Zymo Research). Only samples with an RNA integrity number (RIN) >8 were used. Libraries were generated from 250 ng RNA using TruSeq Stranded Total RNA Sample Prep Kit (Illumina). Paired-end 150-base sequence reads at a sequencing depth of 50 million reads per sample were obtained using Illumina HiSeq 2500 System (Novogene). FASTQ files were analyzed using Strand NGS 4.0 software and aligned to the mm10 mouse genome assembly. Quality of sequencing was ensured by plots for measuring per base sequence quality (Q30 = 99%). Duplicate reads were filtered, followed by quantification and normalization using DESeq method.RNA-seq was performed in triplicates of independent preparations of FLS. SoxCDe novo motif enrichment analysis was performed using MEME-ChIP suite . 10 μg of antibodies against p65 (Active Motif) or SOX4 (Diagenode), coupled to 20 μl Dynabeads (Life Technologies) were used for immunoprecipitating chromatin from each replicate and DNA was purified by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. DNA pooled from the triplicates was used for library preparation. Single-end 50-base sequence reads were obtained at a sequencing depth of 30 million reads per sample using Illumina HiSeq 2500 System and analyzed using ChIP-seq pipeline on Strand NGS 4.0 software. Quality of sequencing was ensured by plots for measuring per base sequence quality (Q30 = 97.8%). Reads were mapped to the mm10 mouse genome build. Peak calling and gene annotation was performed using MACS with an upstream padding distance of 50kb. IP suite .Wildtype mouse FLS were treated with TNF (10ng/mL) for 16 h. Nuclear extracts were prepared from FLS using NE-PER™ Extraction Reagents (Thermo Scientific) protocol. Immunoprecipitation was carried out by conjugating 5 μg of p65 antibody (Active Motif) or rabbit non-immune IgG (Millipore) to 20 μl Dynabeads (Life Technologies). Cell extracts were added to the conjugated beads overnight and eluted protein was analyzed by western blotting using antibodies against SOX4 (Diagenode), p65  or HDAC1.Sox4, Sox11, Il15 and Mapk1 were amplified from mouse genomic DNA and cloned into pBV- Luc, a luciferase reporter plasmid with a minimal promoter and very low basal activity  are functionally redundant and possesses a highly identical HMG-box domain allowing them bind to the same SOX-binding sites on DNA , chromatin remodeling factors such as histone deacetylase 4 (Hdac4), lysine acetyltransferase 6b (Katb6), SET-binding protein 1 (Setbp1) and nuclear receptor coactivator 1 (Ncoa1) and Activating protein-1 (AP-1) signaling components such as mitogen-activated protein kinase 1 (Mapk1/ERK2), mitogen-activated protein kinase 8 (Map3k8/MEKK) and JunB proto-oncogene (Junb/AP-1)  . We founIl15 and Mapk1 we showed the extent of overlap between RELA and SOX4 peaks  resulted in a loss of SOXC-dependent activation of the Il15-reporter in the absence and presence of TNF. Similarly, examples of genes assigned to RELA only peaks such as Sequestosome 1 (Sqstm1), a ubiquitin binding protein that plays a role in autophagy and NF-κB inhibitor beta (Nfkbib), critical intermediate in the canonical NF-κB signaling were upregulated by TNF only in the presence of SoxC genes, but the neighboring gene Ccer remained unchanged by TNF or SoxC knockout. Mgatb4 remained unexpressed in the FLS  TNF activates an autoregulatory switch, which results in shift from RELA binding to SOX4 binding at the Sox4 and Sox11 regulatory regions.In this study we utilized genome wide ChIP-seq and RNA-seq approaches to show that (1) The SOXC family member, SOX4 interacts with RELA in FLS. (2) The SOX4-RELA interaction is likely fortified by the proximity of SOX4 and RELA binding sites on the chromatin. (3) A significant subset of the TNF-responsive RELA target genes require SoxC genes regulate the expression of TNF target genes via multiple different modes of action  belong to Class-1 that require RELA-SOX4 co-binding for their differential regulation. None-the-less the Class-1 genes possess the potential to induce drastic changes in the FLS transcriptome. For instance, the downregulation of histone methylation writer, Kat6b and upregulation of histone acetylation eraser, Hdac4 could potentially remodel the chromatin landscape in the FLS. Overexpression of HDAC4 histone deacetylase correlates with decreased production of proinflammatory cytokines in FLS , were in proximity of a RELA binding event. However, RNA-seq results showed that most of the SOX4-RELA or RELA binding events did not lead to differential gene expression. This suggests that additional factors or stimuli are needed for their expression. Based on the s in FLS  and decrs in FLS . HDAC4 wsynovium , which mMapk1 (ERK2), Map3k8 (MEKK8) and Junb (AP1) is also expected to contribute to large-scale cellular and molecular changes. Regarding ERK2, Wang et al. reported that overexpression of this kinase was involved in maintaining cartilage homeostasis through modulating the TGF-β/SMAD axis via reducing the expression of Col2a1, Aggrecan, and Sox9, which are key molecules for cartilage homeostasis  signaling and that SOX4 expression was required for TGFβ-mediated induction of a subset of SMAD3/SOX4-co-bound genes . Here weSOX2 and SOX9  in the untranslated region (UTR) lead to osteoporosis susceptibility, suggesting an important role for the 3’ UTR in the regulation of SOX4 gene expression in other diseases associated with inflammation  and Startup funds from the Department of Orthopaedics, Emory University School of Medicine to PB.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Arabidopsis thaliana L. and Oryza sativa L. The ZF-HD family members play important roles in plant growth and development, but their contribution to the regulation of plant architecture remains largely unknown due to their functional redundancy. To understand the gene regulatory network controlled by ZF-HD TFs, we analyzed multiple loss-of-function mutants of ZF-HD TFs in Arabidopsis that exhibited morphological abnormalities in branching and flowering architecture. We found that ZF-HD TFs, especially HB34, negatively regulate the expression of miR157 and positively regulate SQUAMOSA PROMOTER BINDING–LIKE 10 (SPL10), a target of miR157. Genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) analysis revealed that miR157D and SPL10 are direct targets of HB34, creating a feed-forward loop that constitutes a robust miRNA regulatory module. Network motif analysis contains overrepresented coherent type IV feedforward motifs in the amiR zf-HD and hbq mutant background. This finding indicates that miRNA-mediated ZF-HD feedforward modules modify branching and inflorescence architecture in Arabidopsis. Taken together, these findings reveal a guiding role of ZF-HD TFs in the regulatory network module and demonstrate its role in plant architecture in Arabidopsis.In plants, vegetative and reproductive development are associated with agronomically important traits that contribute to grain yield and biomass. Zinc finger homeodomain (ZF-HD) transcription factors (TFs) constitute a relatively small gene family that has been studied in several model plants, including  SHOOT MERISTEMLESS (STM), REVOLUTA (REV), and CUP SHAPED COTYLEDONS (CUC) 1, 2, and 3, are class I KNOTTED-like homeobox TFs containing a StAR-related lipid-transfer lipid-binding domain, an HD-ZIP domain, and a No Apical Meristem NAM domain, respectively  ethanesulfonic acid (MES) , pH 5.5, 10 mM MgSO4), and then used to inoculate the abaxial epidermal side of 3–4-week-old N. benthamiana leaves. Three days after infiltration, GFP signals in leaves were observed on an LSM710 confocal microscope .Each TF gene (without a stop codon) was cloned into vector pMDC83 harboring the 35S promoter using LR Clonase. TF coding regions were C-terminally fused to GFP, and the fusion proteins were expressed under the control of the CaMV 35S promoter using the Gateway-Compatible Plant Transformation vector . The resArabidopsis was extracted using TRIzol reagent . RNA samples were treated with DNase I using the TURBO DNA-free kit . cDNA was synthesized from 2 µg of total RNA using the Superscript III First Strand Synthesis kit . Each primer was designed to amplify a 200–300 bp region of the corresponding transcript, and the UBI10 transcript was used as the normalization control for qPCR. All real-time PCR analyses were performed on a C1000 Thermal Cycler . Primers were tested on WT Col-0 by quantitative PCR using the Bio-Rad CFX96 Real-Time System to check primer quality, as determined by melting curve analysis and amplification efficiency. Gene expression was measured in three technical replicates for each sample; for these experiments, cDNA was generated by reverse transcription of 2 µg of total RNA, as described above. Primer sequences used for qPCR are provided in For real-time qPCR, total RNA from the leaves and young floral tissues of zf-hd mutants. Primers were designed using UPL RT primer [To determine the effects of miRNAs, we used the stem-loop reverse-transcription protocol (TaqMan assay) to compare mature miRNA expression levels between wild type and T primer . snoR41 www.arabidopsis.org, accessed on 5 June 2014) was used as the reference genome, and the TAIR10 genome annotation was used as the gene model with no novel junctions. For differential expression analysis on WT flower and mutant flower tissues, DESeq was used with default parameters [http://www-huber.embl.de/users/anders/HTSeq/doc/count.html), using the chromosomal coordinates of Arabidopsis thaliana microRNAs from miRBase (http://www.mirbase.org) v19 as the model and option “-m intersection-nonempty”. Library size was normalized against the total number of 18–30-nt small RNA reads in each library. Differential expression of mature miRNAs and precursors were analyzed using DESeq with default parameters [Using the TRIzol method, total RNA was isolated from WT and mutant flower tissues and roots from plants grown in a greenhouse or growth chamber. RNA quality was checked on an Agilent Technologies 2100 Bioanalyzer, and RNA-seq libraries were prepared using the Illumina TruSeq RNA Sample Prep kit . Small RNA libraries were prepared following the TruSeq Small RNA Library Prep Kit . Libraries were generated by Macrogen, and sequencing was performed on the HiSeq 2000 platform . Expression analysis of roots and flower tissues was evaluated using Tophat for mapping and Cuffdiff for differential expression analysis, both with default parameters ,60. The rameters . For smarameters . Read corameters . GO termrameters .35S:HB34-GFP, and confirmed the expression of HB34-GFP  from 3 kb upstream of a transcription start site (TSS) or 1 kb downstream of a transcriptional termination site (TTS) were identified as candidate HB34 targets.For ChIP-seq, we constructed transgenic plants expressing HB34 fused to GFP expressed under the control of the 35S CaMV promoter, HB34-GFP . A homozhttp://neomorph.salk.edu/PlantCistromeDB) [p-value < 0.001).We derived the top PWM from the experimental data (ChIP-seq and RNA-seq) using MEME-ChiP . In additromeDB) . We usedtromeDB) . To detep-values for the FFL types, we shuffled the expression values of the miRNAs and target genes 10,000 times and compared the randomized FFL distributions.We identified four types of FFLs involving the ZF-HD TF HB34 by combining our expression data with miRNA targets obtained from a literature review and psRNATarget . In our SPL10. We found that ZF-HD TFs, especially HB34, negatively regulate the expression of miR157 and positively regulate SPL10. Genome-wide ChIP-Seq analysis revealed that miR157D and SPL10 are direct targets of HB34, creating a feed-forward loop that constitutes a robust miRNA regulatory module. Network motif analysis contained overrepresented coherent type IV feedforward motifs in the amiR zf-HD and hbq mutant background. This finding indicates that miRNA mediated ZF-HD feedforward modules modify branching and inflorescence architecture in Arabidopsis. Taken together, these findings not only reveal a guiding role of ZF-HD TFs in the regulatory network module but also demonstrate its role in plant architecture in Arabidopsis. Furthermore, the model herein provides insights into candidate genes that determine agronomic traits associated with yield.Our data points to a novel feed-forward loop between a ZF-HD, miR157, and its target genes, in which loss of function leads to increased branching and inflorescence architecture. In this study, we identified a new function of ZF-HD34 (HB34): direct regulations of miRNA157D and its target


USP22 gene amplification seldom occurs, and the mechanism underlying USP22 upregulation in human cancers remains largely unknown.Ubiquitin-specific protease 22 (USP22), a putative cancer stem cell marker, is frequently upregulated in cancers, and USP22 overexpression is associated with aggressive growth, metastasis, and therapy resistance in various human cancers including lung cancer. However, USP22 gene was selectively constructed to screen against a customized siRNA library targeting 89 selected transcription factors to identify potential transcription factors (TFs) that regulate USP22 expression in human non-small cell lung cancers (NSCLC). Association of identified TFs with USP22 and potential role of the TFs were validated and explored in NSCLC by biological assays and immunohistochemistry analysis.A luciferase reporter driven by a promoter region of Luciferase reporter assays revealed that SP1 and activating transcription factor 3 (ATF3) inhibit USP22 transcription, while transcription factor AP-2 Alpha/Beta (TFAP2A/2B) and c-Myc promote USP22 transcription. Binding site-directed mutagenesis and chromosome immunoprecipitation (ChIP) assays validated AP2α and AP2β are novel TFs of USP22. Furthermore, overexpression of AP2A and AP2B significantly upregulates USP22 expression, and its target: Cyclin D1, concurrently enhances the proliferation, migration, and invasion of NSCLC A549 and H1299 cells in a partially USP22-dependent manner. Moreover, AP2 protein level correlated with USP22 protein in human NSCLC tissues.USP22 gene expression to promote the progression of NSCLC, and further support USP22 as a potential biomarker and therapeutic target for lung cancer.Our findings indicate AP2α and AP2β are important transcription factors driving Video AbstractThe online version contains supplementary material available at 10.1186/s12964-022-00946-9. USP22 gene is located on short arm of the chromosome 17p11 and plays an important role in developmental and cancer biology through both histone ubiquitination dependent and independent pathways [” 11-gene signature associated with poor prognosis in a variety of cancers [, in vivo angiogenesis, growth, and metastasis of KRAS mutant lung cancer, and significantly sensitized these cells to cisplatin and irradiation independent p53 status [Ubiquitin-specific protease 22 (USP22) is a histone-modifying enzyme whose predominant function is the removal of the mono-ubiquitin moiety from lysine 120 of H2B (H2Bub1) [pathways . In canc cancers . USP22 i cancers –7. USP22 cancers , 8, whil cancers , 10. Mec cancers . USP22 k3 status –16.USP22 somatic mutations is very low, and gene sequencing and mRNA expression data reveal that USP22 gene is more frequently deleted or under-expressed than amplified or overexpressed in those same cancer types for which IHC studies found USP22 to be overexpressed [Regulation of USP22 expression is still poorly understood in cancer. The widely accepted finding of USP22 overexpression in cancer by immunohistochemistry (IHC) analysis is not consistent with the results of several additional genome-wide studies . At the xpressed –19, indixpressed  and cAMPxpressed  regulatexpressed . At the xpressed .USP22 gene with a panel of gene-specific siRNAs targeting these selected transcription factors (TFs) to identify potential TFs regulating USP22 expression in human NSCLC cells. We found that transcription factor 2 , c-Myc, and nuclear transcription factor Y subunit alpha (NF-YA) promote the transcription of USP22 gene, while SP1 and activating transcription factor 3 (ATF3), a member of the ATF/CREB family of transcription factors, which may represent a potential tumor suppressor in lung cancer [USP22 gene in NSCLC cells. Given that the role of AP2 in USP22 expression has not been previously described, we further focused on regulation of USP22 by AP2. AP2 consists of five paralogs in mammals; AP2A to AP2E belonging to the AP2 transcription factor family [USP22 gene expression; and revealed an association of AP2 with USP22 in lung cancer tissues, suggesting AP2 may promotes the progression of lung adenocarcinoma partially via enhancing USP22 expression.Here, we investigated the underlying mechanism of USP22 expression in NSCLC using luciferase reporters driven by promoter regions of g cancer , inhibitr family . In addir family . In thisThis study was reviewed and approved by the Institutional Review Board (IRB) of City of Hope National Medical Center. A cohort of 240 NSCLC patients who underwent surgical resection with for curative intent between 2002 and 2014 without preoperative chemotherapy or radiation therapy were included, and patients’ clinical characteristics were reported previously [USP22 knockout (USP22−/−) cells were generated from H1299 and A549 cell lines by CRISPR/Cas 9 system as previously described [Human lung adenocarcinoma cell lines H1299 and A549 were purchased from ATCC. The H1299 cells were cultured in RPMI 1640 and A549 cells were cultured in DMEM supplemented with 10% FBS and 100U/ml penicillin and streptomycin. Both cells were authenticated by the Integrative Genomics Core by using short-tandem repeat polymorphism analysis before use. Cell lines were tested mycoplasma free using colorimetric mycoplasma detection assay with HEK-Blue-2 cells as described before . USP22 kescribed .USP22 gene was cloned and constructed into PGL 4.14 vector by Kpn I and Xho I (NEB). Other deletion fragments were also cloned into PGL4.14 and sequences were acquired by PCR using the -5085/ + 55 plasmid as a template. Primers are shown in Additional file Genomic DNA was isolated from H1299 cells using DNA extraction Kit (QIAGEN). The promoter region from -5085 to + 55 of the USP22 promoter-driven luciferase reporter plasmids and Renilla-luciferase expressing plasmid in a 10:1 ratio by using lipofectamine 3000. For siRNA screening, 3000 cells for each well were mixed with transfection master mix, and then seeded in 96 well plates. Forty-eight hours after transfection, cells were washed with PBS and lysed for 15 min in room temperature. Supernatant was then analyzed by TECAN plate reader for both Firefly and Renilla luciferase activity according to the manufacturer’s instruction of Dual-luciferase reporter system . The list of siRNA library targeting for transcription factors was shown in Additional file H1299 and A549 cells were plated in plates 24 h before being transfected with Cells were seeded in 6-well plates and transfected with siRNA or plasmids. Cell proliferation, migration and invasion assay were performed as described previously .Total cellular protein was extracted with SDS lysis buffer and heated in 95 °C for 7 min. Electrophoresis was performed and proteins were transferred onto PVDF membranes using Thermo Fisher i-Blot machine. Membrane was blocked by 5% milk in PBST for 1 h at room temperature and was incubated with primary antibody overnight. After washing out the primary antibody, the membrane was incubated with a secondary antibody for 1 h and sent for imaging. A primary antibody against β-actin (1:3000), c-Myc, AP2A, AP2B, and FLAG-HRP was purchased from Santa Cruz Biotechnology. Primary antibody against USP22 was purchased from Abcam. Antibody against cyclin D1 was purchased from Cell Signaling Technology.7 cells were cultured in 10-cm plate. Cells were fixed by adding formaldehyde to a final concentration of 1% for 10 min and glycine was added to neutralize untreated formaldehyde. Cells were washed, lysed and collected in 1.5 ml tube and sonicated under 4 °C to shear the cross-linked DNA to 200–1000 bp. The cross-linked protein-DNA was immunoprecipitated with magnetic beads, and a combination of anti-AP2α and AP2β antibodies (these two TFs bind to the same DNA sequence), and a normal IgG as a control for nonspecific binding at 4 °C overnight with rotation. The beads containing protein-DNA complex were pulled down by magnet holder. After several washing steps, the purified protein-DNA complexes were eluted. The cross-link of protein-DNA complexes were reversed by 5-h incubation and rotation at 65 °C with proteinase K. qPCR was performed using primers for the USP22 promoter region. Primers used for qPCR are: forward primer: 5’- CGACACCCTCCGCGAACC -3’ and reverse primer: 5’- CGGAGGCCGGACAAAGAT -3’.ChIP assays were performed using EZ-ChIP Kit (Millipore) according to manufacturer’s instructions. 1 × 10https://github.com/IndrajeetPatil/ggstatsplot).The Cancer Genome Atlas (TCGA) lung cancer Illumina HiSeq normalized RNA sequencing  data were downloaded from the University of California Santa Cruz Xena Browser (xenabrowser.net/datapages/) for correlation expression analysis by running the ggscatterstats function of the R package ggstatsplot . Statistical analysis was performed using Student’s t-test for group–group analysis and Spearman correlation was performed for correlation studies.USP22 gene, we uploaded the sequenced −5261/+55 region in human USP22 promoter to PROMO website [USP22 promoter, and about 136 TFs including the reported SP1 and CREB  was critical to USP22 transcriptional activity. As shown in Fig. To characterize TFs that regulate expression of  website  to prediUSP22 promoter region; a customized siRNA library (Themo Fisher) including 89 validated siRNAs targeting known TFs and factors potentially binding to the region  was generated  and A549 (lower panel) cells, and the results showed that two individual specific siRNAs targeting AP2A (left panel) and AP2Β (right panel) markedly decreased mRNA expression , is an important negative regulator of USP22 transcription. Similarly, in this study, we also found critical transcriptional inhibitors may be present in − 185 to + 55, and the data from the luciferase reporter assay also indicated that potential transcription factors binding to other sites outside of this region may promote USP22 transcription. We found that there are several binding sites of AP2 transcription factor in the USP22 promoter region, and an AP2 binding site close to upstream of the USP22 transcriptional start site (− 12 to − 3) that almost overlaps with the SP1 binding site, is critical to enhance USP22 transcription. We found that mutation or deletion of these motifs, particularly those closest to the translational start site, resulted in a significant decrease in USP22 promoter activity, suggesting that the AP2 promotes USP22 gene transcription.In the study, by bioinformatics analysis, we found that within the region from -3296 to + 55 of tors SP1 , CREB1 [tors SP1 , and HIFtors SP1  regulatetors SP1 . SurprisConsistent with the potential oncogenic role of USP22 in lung cancer, AP2 also appears to play a positive role in tumorigenesis. Therefore, loss of activity of AP2 can lead to the impairment of proliferation, differentiation and apoptotic processes, suggesting AP2 activity may play a role in development . TFAP2A USP22 gene expression, and it seems ATF3 may possess an opposite transcriptional role of the previously reported CREB1 in USP22 expression [Besides AP2, we found that ATF3, a member of CREB protein family of transcription factors may suppress pression . Interespression . In addipression . These opression . TherefoUSP22 mRNA occurred quickly, but lasted only a short time after stimulation or modulation. In addition, APC/C was reported to promote degradation of USP22 during the cell cycle [At the posttranscriptional level, several studies revealed the regulation of USP22 protein by other signals or proteins. We previously showed that cisplatin upregulates USP22 protein in lung cancer stem cells , indicatll cycle .USP22 transcription. Given the important roles of both USP22 and AP2 in lung cancer carcinogenesis, our finding of the association between these two proteins further supports USP22 deubiquitinate as a potential biomarker and therapeutic target for lung cancer.In summary, our results demonstrate that USP22 expression is promoted by AP2 and c-Myc and is suppressed by SP1 and ATF3 at the transcriptional level. AP2 is an important transcription factor driving USP22 expression, and it may enhance malignancy of NSCLC partially through upregulating Additional file 1. Supplementary figures and tables.


Many transposable elements (TEs) contain transcription factor binding sites and are implicated as potential regulatory elements. However, TEs are rarely functionally tested for regulatory activity, which in turn limits our understanding of how TE regulatory activity has evolved. We systematically tested the human LTR18A subfamily for regulatory activity using massively parallel reporter assay (MPRA) and found AP-1- and CEBP-related binding motifs as drivers of enhancer activity. Functional analysis of evolutionarily reconstructed ancestral sequences revealed that LTR18A elements have generally lost regulatory activity over time through sequence changes, with the largest effects occurring owing to mutations in the AP-1 and CEBP motifs. We observed that the two motifs are conserved at higher rates than expected based on neutral evolution. Finally, we identified LTR18A elements as potential enhancers in the human genome, primarily in epithelial cells. Together, our results provide a model for the origin, evolution, and co-option of TE-derived regulatory elements. MPRAs hTransposable elements (TEs) are repetitive DNA elements that represent a rich source of genetic material for regulatory innovation . In mammcis-regulatory elements. One intrinsic limitation for evolutionary studies is that each enhancer has one ortholog per species barring duplication or deletion, which constrains the sample size for analysis. Within a TE subfamily, each TE is descended from a common ancestor, with each copy evolving mostly independently. This provides a large sample size to draw upon within even a single genome. To serve as a representative subfamily, we selected LTR18A, which we previously identified to be enriched for MAF BZIP transcription factor K (MAFK) transcription factor binding peaks and motifs . Following these criteria, 181 out of 198 LTR18A elements annotated by RepeatMasker are retained . Next, we found primate orthologs for each hg19 LTR18A element by using synteny .From our set of manually curated human LTR18A elements and their orthologs, we computationally reconstructed the LTR18A phylogenetic tree using a two-step process. Based on the unique characteristic of TEs to multiply by transposition and the presence of orthologous copies in different primate genomes, we split our reconstruction of LTR18A evolution into two phases corresponding to transposition and speciation A. For eaSupplemental Fig. S1). One feature of note occurs in node 43, two nodes from the root of the tree . In hg19, 13/181 elements contain the insert. The insert-containing elements are spread throughout most of the hg19 LTR18A phylogenetic tree, which is consistent with a deep ancestral origin for the insert and occurrence in node 43 of our reconstruction. Additionally, we found that the CEBP motif is in the LTR18A consensus and enriched in the subfamily relative to genomic background (DBP log odds ratio 6.5). If the CEBP motif is functionally important, the insertion of a second CEBP motif could be an ancestral gain-of-function mutation. In conclusion, our reconstruction is able to generate a subfamily ancestor similar to the Repbase consensus and reveals evolutionary events that would otherwise be missed.Next, we evaluated our reconstructed LTR18A sequences to see if they are consistent with those derived from other methods. TE consensus sequences are often used as a representation of the ancestral state of the subfamily. Excluding insertions and deletions, our reconstructed LTR18A subfamily ancestor has an ∼5.9% substitution rate relative to the LTR18A consensus sequence, which is lower than the 16.1% subfamily average. This suggests that although we start from different elements and use different methodologies, both our reconstruction and the Repbase consensus are approaching each other. In addition to substitutions, our reconstructed ancestor also has ∼8.0% insertions compared with the consensus. The insertions appear to be caused by the consensus dropping bases if the majority of elements do not have the base in the alignment, as well as PRANK's tendency to include insertions when alignable sequence is present in more than one element. The MAFK motif enriched in LTR18A was present in both our reconstructed subfamily ancestor and the Repbase consensus. Overall, the topology of our reconstructed phylogenetic tree resembles the tree generated from all hg19 LTR18A elements  . In one 2 of the RNA over DNA ratio followed by normalization to the basal hsp68 promoter. Normalizing to the basal promoter allowed us to have the same reference point between cell lines. Active elements were defined as those with enrichment scores greater than one, representing elements that increase transcription by more than twofold. When we compare the distribution of enrichment scores for HepG2 and K562, we find that LTR18A elements are generally more active in HepG2 than K562, which is consistent with cell type–specific activity commonly seen in enhancers (P < 2.2 × 10−16) , implying differential sequence features required for enhancer activity between cell lines.To understand cell type effects, we tested LTR18A for enhancer activity in HepG2 and K562 cell lines. We calculated enrichment scores for each element by taking the lognhancers A. Out ofTo identify important sequence features for enhancer activity, we took advantage of the natural sequence variation within LTR18A elements. Using AME motif enrichment analysis , we aske2 ratio of each motif-mutated LTR18A sequence to its native sequence . Across LTR18A elements of our reconstructed phylogenetic tree, we were able to confirm that regions of increased SHARPR nucleotide activity were enriched for CEBP and AP-1 motifs . As SHARPR nucleotide activity scores could discover the same biologically meaningful sequences as our previous analyses, we took the sum of activity scores across each LTR18A element and annotated them in our tree .One of our primary goals was to understand how enhancer activity of LTR18A as a subfamily changed over time. To address this question, we synthesized 160-bp tiles at 10-bp intervals across each LTR18A ancestral sequence, ortholog ancestor, and hg19 element used in reconstruction B. After our tree A. From aour tree C. This lour tree B. On theour tree D. FinallTo further investigate why enhancer activity changes in our LTR18A tree, we looked at differences in CEBP and AP-1 motif presence using DBP and JUN as representatives. When elements are categorized by the number of DBP and JUN motifs, the number of motifs is positively correlated with SHARPR sum E. FurtheSupplemental Table S2). The increased substitution rate holds true even when only considering masked regions of the genome. Although it is possible that the genomic background rate includes regions under selection, the LTR18A substitution rates across primate species are overall inconsistent with purifying selection for the subfamily. Furthermore, both phyloP and phastCons scores at LTR18A elements provide no evidence of selection at the subfamily level across 30 mammals, including 27 primates  than the corresponding genomes  of all 10-bp windows are significantly more conserved than the average window. The majority (84%) of conserved 10-bp sliding windows are shared across all seven primates for a total of 24.5% (85/347) possible 10-bp windows covering 58% of the LTR18A consensus (208/357 bp) being classified as conserved. Shared, conserved regions defined by our sliding window analysis contain transcription factor motifs, including AP-1 and CEBP A.Because CEBP and AP-1 motifs are critical for enhancer activity, we hypothesized that the motifs provided by LTR18A have been under purifying selection and, consequently, show higher conservation than expected under a neutral model of evolution. To obtain the background motif conservation rates, we adapted a method previously used in yeast . BrieflySupplemental Fig. S9). Altogether, the overall lack of active epigenetic marks at LTR18A in HepG2 and K562 implies that they are largely inactive as regulatory elements in the two cell lines, despite many showing enhancer activities in reporter gene assays. We hypothesized that epigenetic repression of LTR18A may be the cause for the lack of active enhancer marks in HepG2. Consistent with this hypothesis, repressive histone mark H3K9me3 is enriched over LTR18A compared with the surrounding genomic region, with the peak in signal possibly indicating that LTR18A is targeted for silencing . These results suggest that although LTR18A elements possess the sequence features necessary for enhancer activity, they can be epigenetically silenced.Our MPRA reveals that LTR18A elements have the sequence features to be activating regulatory elements depending on cellular context. To explore the relationship between regulatory potential from MPRA and enhancer function in the genome, we examined epigenetic marks in HepG2 and K562 using ENCODE data . We firscis-regulatory elements (cCREs) as defined by ENCODE Registry V2 across 839 cell/tissue types . Among fully classified cell/tissue types, keratinocytes have the highest number of LTR18A elements associated with ELS, followed by the PC-3 and PC-9 cell lines . This is perhaps unsurprising, as transcription is typically the first step of transposition and provides the substrate for integration of retrotransposons. However, one important consequence is that transcription factor binding sites that contribute to TE regulatory potential could be enriched within a subfamily owing to biased lineage amplification. This appears to have been the case for the recently reclassified LTR7 subfamilies, each of which possess a unique set of transcription factor motifs and underwent a wave of genomic expansion to fill different early embryonic niches  of all elements. On the other hand, the major lineage of LTR18A has enhancer activity throughout transposition. The stark contrast between the two lineages in enhancer activity and abundance leads us to speculate that the regulatory potential of LTR18A was directly related to its ability to expand in the genome, a hypothesis with which our data are consistent  in length. Finally, both elements of a pair need to pass all requirements to be selected for ancestral reconstruction. Orthologs were defined using the chain files from UCSC to find LTR18A elements within the same syntenic blocks . LTR18A Supplemental Methods). After manual curation of ortholog ancestors, we used MAFFT and PRANK to reconstruct the phylogenetic tree and sequences of LTR18A subfamily ancestral sequences.Ancestral reconstruction of both ortholog ancestors and subfamily ancestors used MAFFT and PRANK followed by manual curation . To geneSupplemental Fig. S4), 37 pairs of elements for JUN mutagenesis, and 46 pairs of elements for DBP mutagenesis. Elements for mutagenesis were chosen based on the closest primate ortholog with/without the motif. JUN motifs were mutated to TCACCAATGGT, and DBP motifs were mutated to TCCCACAGCAT. Non-motif-containing elements were mutated to GCTGAGTCATG for JUN and ATTATGTAACC for DBP. We also made DBP and JUN mutations in ancestral node 45 and 43 and the Repbase consensus, resulting in seven additional mutated motif-focused and 168 tiled sequences. For positive and negative controls, we selected 223 regions from a previous study by Ernst et al. . Expression of an element was calculated as RNA CPM/DNA CPM. Expression was normalized to the average of basal construct transfection replicates. Finally, enrichment score was calculated as the logE-value threshold of 0.05  in <20% of all LTR18A sequences were removed. This filter retained all consensus base positions.P-value < 5 × 10−5.GWAS analysis tool PLINK was used to identify nucleotides significantly associated with the phenotype, such as MPRA activity/inactivity or ATAC peak . We limit-test and more than significant nucleotides within the motif.From the list of significant nucleotides in TE-WAS, we identified transcription factor motifs from the core human HOCOMOCOv11 database that are overrepresented based on information content . InformaFrom tiled MPRA, we calculated regulatory activity for full-length elements using SHARPR with a few adjustments . For eacTo validate the SHARPR approach, we identified motifs that were enriched in peaks, or regions of high nucleotide activity. Peaks were defined as regions with nucleotide activity scores greater than three standard deviations above the basal mean. Enriched motifs were then identified in peak regions using AME using shuffled sequence as background .t-test comparing conservation across elements in the window against conservation across all windows, with a P-value threshold of 0.05 after Bonferroni correction. Only windows that were conserved in all seven primate species were kept for further analysis. Motif scanning by FIMO was performed to find transcription factor motifs fully within conserved windows  were excluded. The mean conservation was then calculated for each 10-bp window separately for each species. Windows were determined to be significantly conserved using  windows .k-mers of the motif length that are found by FIMO   . The phyect.org) . Overlapect.org) . Enrichmect.org) . LTR18A P-values below 0.05 after correcting for number of motifs tested were considered significant. The top six cell/tissue types were selected for analysis as they provided the greatest number of LTR18A elements overlapping cCREs.Fisher's exact test was used to determine if transcription factor binding motifs in LTR18A elements are associated with cCRE overlap. Motifs that had https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE201068. All custom scripts are available in Supplemental Code.All raw and processed sequencing data generated in this study have been submitted to the NCBI Gene Expression Omnibus (GEO;


Transcriptional switches can be utilized for many purposes in synthetic biology, including the assembly of complex genetic circuits to achieve sophisticated cellular systems and the construction of biosensors for real-time monitoring of intracellular metabolite concentrations. Although to date such switches have mainly been developed in prokaryotes, those for eukaryotes are increasingly being reported as both rational and random engineering technologies mature. In this review, we describe yeast transcriptional switches with different modes of action and how to alter their properties. We also discuss directed evolution technologies for the rapid and robust construction of yeast transcriptional switches. The field of synthetic biology has led to the construction of increasingly sophisticated biological systems ,2, incluEndogenous systems, such as the Gal4 transcriptional switch ,7,8 and Unfortunately, most genetic switches cannot be used directly for synthetic biology applications because of their inappropriate switching properties. For example, such promoters often exhibit detectable activity in the OFF-state (leaky expression), which hampers the regulated high-level expression of toxic proteins . FurtherSaccharomyces cerevisiae.In the present review, we summarize how synthetic transcription-level genetic switches have been created and improved in yeast. First, we categorize yeast synthetic transcription switches into two groups according to the mode of action: a transcription activation mode A,B, and Synthetic genetic switches in yeast are generally categorized according to two types of regulation . One is Although transcription from a yeast promoter requires the recruitment of multiple endogenous TFs to the promoter, the binding of a single protein fused with eukaryotic transcription activators (eTAs) is sufficient to artificially stimulate the recruitment of yeast TFs. Therefore, synthetic transcriptional switches can be created in yeast by fusing eTAs with ligand-responsive DNA binding proteins, such as bacterial TFs . Using appropriate design parameters, ligand-dependent binding of bTFs to their operator DNA sequences fused upstream of the yeast core promoter  can be translated to the output gene expression ,27,28,29tetO) fused upstream with a eukaryotic core promoter based on a yeast CYC1 promoter which loses its UAS in response to doxycycline, a more effective analog of tetracycline . In. InS. ceectively . In subsectively .When an intact bacterial transcription repressor binding sequence is fused to a position upstream of the TATA box in a yeast promoter, transcription is blocked by inhibition of RNAP complex binding. Transcription from the resultant synP is derepressed when the ligand-bound bTFs dissociate from the synP. This mode of action has allowed the creation of many inducible yeast promoters.tetO, called Tet-ON, in which intact TetR binds to tetO and hinders the binding of RNAP to the promoter (transcription repression)  fused to the FapR repressor in terms of their activation capacities  . They fobenO and pcaO for BenM and PcaQ, respectively) at single-nucleotide resolution in S. cerevisiae  , antibiovice versa. In most cases, even low expression levels of ON and OFF selection markers are sufficient to allow cell growth and to cause cell death, respectively, which results in low selection efficiency. Moreover, genetic selection fails to discriminate desired genetic switches from others when their output levels under selection are too far from the given selection threshold [Despite the successes of evolutionary strategies described above, this strategy has not been widely applied to genetic switches in yeast. As previously described , stringehreshold ,104. ThuOne possible solution to this problem would involve performing multiple selections for different selection pressures in parallel and choosing the most promising selection pools from which the improved mutants would be screened . We recently described an evolutionary platform for yeast genetic switches that could meet this demand using a trifunctional fusion protein consisting of hsvTK, Zeocin-resistance protein (Ble), and GFP . This fuAs described in this review, a number of bacterial transcription factors (TFs) can easily be assembled into yeast genetic switches with or without the use of an evolutionary strategy. This method is becoming the gold-standard for the engineering of genetic switches in yeast; however, rational engineering for the functional tuning of each component, which is crucial for maximizing genetic switch performance, is not currently feasible. An evolutionary platform for genetic switches in yeast was recently demonstrated ,45,66 ancis,cis-muconic acid production in yeast uses the evolved sensory protein BenM [Genetic switches in yeast that can sense intracellular metabolites, i.e., metabolite sensors, are beginning to be used in the high-throughput screening of higher metabolite-producing cells ,118 and ein BenM . Furtherein BenM ,122,123.