Bdnf) consists of nine non-coding exons driven by unique promoters, leading to the expression of nine Bdnf transcripts that play different roles in various brain regions and physiological stages. In this manuscript, we present a comprehensive overview of the molecular regulation and structural characteristics of the multiple Bdnf promoters, along with a summary of the current knowledge on the cellular and physiological functions of the distinct Bdnf transcripts produced by these promoters. Specifically, we summarized the role of Bdnf transcripts in psychiatric disorders, including schizophrenia and anxiety, as well as the cognitive functions associated with specific Bdnf promoters. Moreover, we examine the involvement of different Bdnf promoters in various aspects of metabolism. Finally, we propose future research directions that will enhance our understanding of the complex functions of Bdnf and its diverse promoters.The gene encoding brain-derived neurotrophic factor ( Bdnf is a eukaryotic gene with a unique genomic structure that is very different from the majority of other genes. It consists of multiple 5′ non-coding exons, also known as 5′untranslated regions (5′-UTRs), and one 3′ exon that codes for the pre-pro-BDNF protein. In rodents, nine functional promoters have been identified upstream of the nine non-coding exons, numbered I to IX or e1 to e9. Promoters I to VIII initiate transcription from the 5′ non-coding Exons I to VIII, which are spliced onto the common coding exon (Exon IX) to generate eight mRNA transcripts. Promoter IX, on the other hand, initiates transcription directly from the 5′-UTR region of the coding Exon IX  generated mouse lines, named Bdnf-e1−/−, e2−/−, e4−/−, and e6−/− mice, in which Bdnf Promoters I, II, IV, and VI were selectively disrupted. Specifically, an enhanced GFP-STOP codon sequence was inserted downstream of a non-coding exon, resulting in the transcription of an exon-GFP-STOP codon-coding sequence RNA and the translation of enhanced GFP instead of Bdnf protein  driven by Bdnf Promoter I) were found in abundance in the cingulate cortex, lateral hypothalamus (LH), piriform cortex [Bdnf-e6 neurons were mostly found in some hippocampal areas and multiple regions in the frontal cortex [Bdnf transcripts remain largely elusive.To investigate the function of specific  protein . These mcterized ,11,23. Bm cortex , basal lm cortex . Bdnf-e6Bdnf in the neurons. Among the nine promoters of Bdnf, the regulatory mechanisms of Promoters I and IV have been the subject of extensive investigation at the molecular level over the past two decades [BDNF plays a crucial role in neuronal development and a wide range of neuronal functions, including neuronal survival, neurite outgrowth, and synaptic plasticity. The biochemical mechanisms underlying the downstream signaling of BDNF and its receptor TrkB have been extensively studied  [Bdnf transcripts from Promoter I, mediated by cAMP, Ca2+, and CREB [Bdnf Promoter I can also be regulated by epigenetic factors. Hara et al. showed that potassium chloride (KCl) induced depolarization, leading to the acetylation of histones H3 and H4, mediated by Ca2+ signals, and resulting in a significant increase in the amount of Promoter I-driven Bdnf transcripts [ (NF-kB) , the cal (NF-kB) , and neu (NF-kB) ,53,54 . This suggests a relationship between Promoter I and dendritic structural plasticity in the piriform neurons [Bdnf-e1 transcripts in different types of neurons and under various physiological and pathological conditions.As of now, the cellular functions of  neurons . FurtherBdnf Promoter II, suggesting a potential role of this promoter as an endogenous target for neuronal repair or regeneration [Similar to Promoter I, Promoter II is an activity-dependent promoter. Fewer molecular and cellular studies have been conducted on this promoter compared with those on Promoters I and IV ,59. Althneration .Bdnf Promoter IV was initially classified as Promoter III until 2007, when several new Bdnf promoters were discovered, leading to a change in the nomenclature [Bdnf Promoter IV contains multiple binding sites for transcriptional factors, including three calcium-responsive elements (CaRE) co-activated by the calcium-response factor (CaRF), USF1/USF2, and CREB [The nclature . Bdnf Prand CREB ,61,62,63and CREB  and an Eand CREB   and increased expression of the serotonin-synthesizing enzyme tryptophan hydroxylase (TPH) and the dopamine D4 receptor (DRD4) [Bdnf is involved in the regulation of monoamine systems. Additionally, Bdnf-e4−/− mice displayed cholinergic dysfunctions in the frontal cortex and hippocampus compared with wild-type mice [Using a et al.  demonstrr (DRD4) . These rype mice .Bdnf-e4 transcripts have also been found in cultured rat cortical astrocytes following extracellular ATP stimulation [In addition to being expressed in the neurons, mulation . Future Bdnf Promoter VI underwent a change in nomenclature in 2007 when several new Bdnf promoters were discovered [Bdnf Promoter VI contains multiple binding sites for transcriptional factors, including two GC boxes and a GA-rich sequence that can both be bound by specificity protein 1 (Sp1) [2+/calmodulin-dependent protein kinase II (CaMKII) [Initially classified as Promoter IV, scovered . Bdnf Pr 1 (Sp1) . It also(CaMKII)   , NGF tre(CaMKII) . Interes(CaMKII) .Bdnf promoters, Promoters I, II, and IV exhibit activity-dependent characteristics. Promoters I and IV share similar regulatory elements, such as PasRE, NFkB-RE, and CRE, which can be activated by the transcriptional factors NPAS4, NFkB, and CREB, respectively  in the genomic structure of Bdnf, so Promoter II may also use the regulatory elements upstream of Promoter I. However, it also exhibits a specific regulatory element downstream of its own transcription start site (TSS), the NRSE. Interestingly, Exon II contains two more alternative splice sites, leading to three different transcripts from Promoter II . However, the specific functions of Transcripts IIa, IIb, and IIc are still unknown. Distinct from Promoters I, II, and IV, Promoter VI has different regulatory elements, such as GC box and CCAAT sequences, which are not responsive to activity.Among the four investigated ectively A. In addAlthough the cellular functions of Promoter IV-driven BDNF have been discovered to contribute to the development of PV interneurons, GABAergic transmission, and synaptic plasticity , the funRecent research has also demonstrated that various metal ions, such as copper, zinc, and lithium, play a significant role in modulating the expression and functions of BDNF . Zinc anBdnf is known to play a critical role in a wide range of cognitive functions and has been implicated in various psychiatric and neurological disorders, such as depression, anxiety, schizophrenia, and neurodegenerative diseases. Over the past decade, numerous reviews have provided comprehensive summary and discussion of this topic [Bdnf transcripts/promoters in different psychiatric disorders and cognitive functions. is topic ,91,92,93Bdnf and mood disorders, particularly depression and anxiety . DespitBDNF Promoter IV may play a role in the development of depression [BDNF gene polymorphism known as the C allele at rs12273363 has been found to be associated with mood disorders in humans, as compared with the more common T allele [Bdnf transcription [A human study has indicated that the pression . To be sT allele . NotablyBdnf transcripts driven by Promoters IV and VI were significantly reduced, while the transcripts driven by Promoters I and II remained unaffected. This reduction could be rescued by chronic treatment with imipramine, a tricyclic antidepressant [Bdnf alone, without the contribution of traditional antidepressants, can rescue depression in socially defeated mice is an unexplored question. To investigate whether dysfunction in the Bdnf Promoter IV gives rise to depression, Sakata et al. [Bdnf-e4−/− mice. The study found increased immobility time in the tail suspension test (TST), less preference for sucrose, and elevated escape latency in the learned helplessness test (LHT) [Bdnf-e4-/- mice, but with no increase in hippocampal Bdnf levels [Bdnf, one cannot rule out the possibility that the antidepressants work through other Bdnf promoters or in brain regions other than the hippocampus.Social defeat is a widely used behavioral paradigm to induce depressive behaviors in mice. Tsankova et al.  found thpressant . Howevera et al.  conductest (LHT) . In anotf levels . While tBdnf-e4−/− mice at an early age induced the secretion of Bdnf in the hippocampus and rescued depressive-like behaviors [Bdnf promoters have been implicated in depressive disorders. Further work studying the relationship between early brain development and Bdnf promoters may reveal the underlying mechanisms.Interestingly, providing an enriched environment to ehaviors . Thus, oBdnf may not be attributed solely to Promoter IV but also to other Bdnf promoters. Some findings have suggested that different antidepressants can activate distinct Bdnf promoters. Specifically, Tsankova et al. demonstrated that imipramine activated Promoters IV and VI, but not I and II [Bdnf-e2 transcripts, while desipramine treatment increased Bdnf-e4 transcripts specifically. However, fluoxetine treatment did not significantly affect all Bdnf transcripts [Bdnf-e1, but not the production of e2 in the rat hippocampus [Bdnf is diverse and complex.The complex relationship between antidepressants and the expression of I and II , whereasI and II  observednscripts . Moreovepocampus . TogetheBDNF in the treatment of MDD. A recent cell culture study reported that fluoxetine and venlafaxine could increase the methylation of BDNF Promoter IV, and the methylation of position CpG-39 could significantly elevate the activity of Promoter IV [BDNF Exon IV in depressed individuals [Bdnf, both dependent and independent of Promoter IV.In a human study, Tadić et al.  providedmoter IV . Interesividuals . FurtherBDNF and schizophrenia, and have identified a well-known BDNF polymorphism, rs6265 , as a potential biomarker for the risk of developing schizophrenia , and BDNF-e6 transcripts were decreased in the hippocampus of schizophrenia patients compared with healthy controls. In another study, Chen et al. [Bdnf-e6−/− mice to investigate the relationship between Bdnf Promoter VI and schizophrenia-like phenotypes. They found that a deficiency in the Promoter VI-driven expression of Bdnf combined with adversity early in life gave rise to schizophrenia-like behaviors, such as deficits in social interactions, spatial memory, and sensorimotor gating, as measured by the pre-pulse inhibition test (PPI) [Bdnf Promoter VI and the risk of developing schizophrenia-like behavior, at least in rodents. In a human study of cohorts of schizophrenia patients in the United States and Australia, Wong et al.  found thn et al.  used Bdnst (PPI) , indicatBDNF single nucleotide polymorphism rs6265  was involved in anxiety-related behaviors. Bdnf Met/Met or Bdnf+/– mice exhibited anxiety-like behaviors that could not be rescued by the antidepressant fluoxetine. Several subsequent studies have further explored the relationship between BDNF and anxiety, including the reviews by [Chen et al.  identifiviews by ,97,98.Bdnf-e4−/− mice showed reduced activity in the open field test (OFT), reduced food intake in the novelty-suppressed feeding test (NSFT), and defective response inhibition in the passive avoidance test (PAT) [Bdnf-e4−/− mice did not exhibit anxiety-like behavior in the light and dark box test (LDB) and elevated plus maze test (EPM) [Bdnf+/– mice. It is possible that defects in other promoters may contribute to anxiety in specific situations. Further research is needed to clarify the mechanisms underlying the relationship between BDNF and anxiety and to identify the specific promoters involved. Sakata et al. reported that st (PAT) , which cst (PAT) , a monoast (EPM) , two relst (EPM) . These fBDNF plays a critical role in cocaine addiction and withdrawal, which are governed by the reward system in the brain  of mice conditioned to cocaine, with no such increase observed in mice treated with cocaine but not conditioned to it. Interestingly, Bdnf-e1 transcripts were found to respond to both cocaine treatment and conditioning [As ircuitry . Notablyircuitry  observeditioning , suggestBDNF Exon IV among smokers. Future studies investigating the specific mechanisms through which nicotine shapes the methylation patterns in Exon IV could help shed light on the mechanism of nicotine addiction.Furthermore, nicotine is another addictive chemical that impacts the health of millions of smokers. Two recent human studies conducted in Germany Germany  and BrazBDNF gene in humans have been associated with various psychiatric phenotypes, including anxiety, mood disorders, and aggressive behaviors  is a neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2) , which iBDNF Promoter IV [BDNF Promoter IV by using chromatin immunoprecipitation [BDNF Exon IV promoter and decreased levels of BDNF mRNA and protein [Several studies have revealed a relationship between MeCP2 and moter IV ,118,119.pitation ,118. Notpitation . In addiBdnf promoters are involved in various brain functions, such as circadian rhythm, sleep, and memory formation. For instance, Berchtold et al. [Bdnf-e1−/− and e2−/− mice. Additionally, BDNF plays a critical role in sleep regulation and is influenced by sleep deprivation [Bdnf-e4−/− mice exhibited a decrease in slow-wave activity during non-rapid eye movement (NREM) sleep [Bdnf Promoter IV appears to control executive functions related to learning and memory. For example, Sakata et al. found that Bdnf-e4−/− mice exhibited impaired spatial memory reversal and contextual memory extinction, which are two executive functions, but retained their spatial memory and contextual learning capacity [Previous studies have shown that specific d et al.  found thrivation . Hill etM) sleep , indicatM) sleep ,122,123 capacity .Bdnf haploinsufficiency in children with Wilms’ tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome is associated with severe early-onset obesity [In mammals, the regulation of energy homeostasis is governed by the balance between energy intake (food consumption) and energy expenditure, which includes basal metabolism, physical activity, adaptive thermogenesis, and food digestion . Several obesity .BDNF is primarily expressed in the central nervous system, and its regulation of peripheral metabolic function is thought to occur via neuronal projections from the brain. Numerous studies have shown that BDNF-expressing neurons, predominantly located in the nuclei of the hypothalamus and medulla, play a crucial role in the maintenance of the energy balance . One parBdnf-e1−/−, e2−/−, e4−/−, and e6−/− mice, in which Bdnf Promoters I, II, IV, and VI were selectively mutated [Bdnf-e1−/−, e2−/− mice were overweight, while Bdnf-e4−/−, e6−/− mice maintained a normal weight, consistent with the findings from another study [Bdnf-e1−/− mice housed in a cage with a transparent divider to avoid aggressive attacks [Bdnf-e1−/− mice under normal conditions. It is possible that partial social isolation in the context of Bdnf-e1 knockout, but not Bdnf-e1 knockout alone, may lead to hyperphagia.It is plausible that different BDNF transcripts may contribute to different aspects of the energy balance. Our group has developed  mutated ,25,38. I mutated , Bdnf-e1er study . Interes attacks . In cont attacks  and Chu  attacks  found noBdnf-e1−/− mice, and the Bdnf-e1-expressing neurons in the lateral hypothalamus (LH) were found to specifically regulate thermogenesis [Bdnf-e2−/− mice exhibited significantly increased food intake, indicating that Bdnf Promoter II, rather than Promoter I, may be involved in the regulation of hyperphagia. They also identified a group of Bdnf-e2 neurons in the ventral medial hypothalamus (VMH) that are crucial for promoting satiety. Based on the studies by You et al. [Bdnf promoters and different aspects of the energy balance, and further investigations focusing on the functions of other promoters in the energy balance would be valuable.Moreover, a deficit in thermogenesis was observed in ogenesis . Additiou et al.  and Chu u et al. , it can Bdnf promoters, as well as a summary of the current knowledge on the cellular and physiological functions of different Bdnf transcripts derived from these promoters. In this article, we have provided a comprehensive overview of the molecular regulations and structural features of multiple Bdnf promoters in specific brain regions or neurons is not yet fully understood. Although some studies have identified that specific brain regions [Bdnf promoters, further investigation is required to elucidate the precise mechanisms responsible for the choice of promoter. Second, the spatial distribution of Bdnf transcripts and their corresponding functions at the cellular level is another important question that needs to be addressed. Although single-cell transcriptomic analyses and emerging bioinformatic tools have provided a more comprehensive map of the distribution of Bdnf transcripts, the underlying regulatory networks are still difficult to interpret without the aid of mathematical modeling. Third, understanding the downstream effects of the expression of specific Bdnf promoters in the neurons and how these promoters contribute to distinct neuronal circuits is also an area of active research. The development of newly engineered genetic lines with specific Bdnf promoter-driven Cre proteins, in conjunction with optogenetic tools and tracing viruses, is expected to provide new insights into the neuronal circuits downstream of the expression of specific Bdnf promoters. Finally, the temporal regulation of the expression of BDNF and the potential role of different promoters in brain development is another area of research that requires further investigation. The development of BDNF sensors coupled with genetically designed Bdnf promoter-driven tools may enable the visualization of the expression and release of BDNF controlled by different promoters during brain development.Despite the significant progress made in this field, several important questions remain to be addressed. First, the mechanism underlying the selective expression of different  regions ,2,12 or  regions ,11,13 caBdnf non-coding promoters has the potential to reveal new insights into the complexity of BDNF’s function and provide novel therapeutic targets for the treatment of neurological disorders. Further investigations are required to fully understand the mechanisms underlying the choice of promoter, the spatial distribution, the downstream effects, and the temporal regulation of the expression of BDNF.In summary, the study of


During meiotic prophase I, spermatocytes must balance transcriptional activation with homologous recombination and chromosome synapsis, biological processes requiring extensive changes to chromatin state. We explored the interplay between chromatin accessibility and transcription through prophase I of mammalian meiosis by measuring genome-wide patterns of chromatin accessibility, nascent transcription, and processed mRNA. We find that Pol II is loaded on chromatin and maintained in a paused state early during prophase I. In later stages, paused Pol II is released in a coordinated transcriptional burst mediated by the transcription factors A-MYB and BRDT, resulting in ~3-fold increase in transcription. Transcriptional activity is temporally and spatially segregated from key steps of meiotic recombination: double strand breaks show evidence of chromatin accessibility earlier during prophase I and at distinct loci from those undergoing transcriptional activation, despite shared chromatin marks. Our findings reveal mechanisms underlying chromatin specialization in either transcription or recombination in meiotic cells. During spermatogenic meiosis, chromatin changes due to transcription, homologous recombination, and chromosome synapsis must be coordinated. Here they show that A-MYC and BRDT regulate release of paused RNA PolII to induce a transcriptional burst during pachytene of prophase I. Meiosis is a critical stage in spermatogenesis during which the genome is first replicated and then halved to produce four haploid gametes. The first and longest stage of meiosis is prophase I, during which dramatic changes in chromatin occur to facilitate the events of synapsis and meiotic recombination2. Upon entry into prophase I, the topoisomerase-like protein SPO11, together with multiple accessory proteins, catalyzes the induction of hundreds of DNA double-strand breaks (DSBs) across the genome3. Of these, 90% will resolve as non-crossovers, while the remaining 10% will resolve as crossovers, which result in the physical connection between two distinct chromosomes4. In mammals, DSB induction initiates the assembly of a tripartite protein structure called the synaptonemal complex (SC), which tethers homologous chromosomes and facilitates the progressive repair of DSBs6. By the end of prophase I, when the SC begins to break down, the DSB-initiated crossovers tether homologous chromosomes until the first meiotic division7. These events, from DSB formation through the first meiotic division, require a complicated and intricate series of changes to chromatin that are essential for both meiotic recombination and the proper segregation of DNA into haploid gametes.Spermatogenesis is a multistep process required for the daily production of millions of spermatozoa within the seminiferous epithelium of the testis8. Prophase I spermatocytes initiate the transcription of thousands of protein-coding genes, pseudogenes, piRNAs, long non-coding RNAs, and transposable elements, establishing a highly complex transcriptional program13. Many of the genes transcribed during this stage are necessary to facilitate meiotic recombination and synapsis during prophase I itself14. In addition, several genes that are critical for spermiogenesis are expressed during prophase I, while others, such as the Y-linked Zfy1 and Zfy2 genes, must be transcriptionally repressed until they are needed to facilitate events during transcriptionally inert stages later in spermiogenesis15. Dysfunctional gene expression during prophase I can lead to a variety of infertility phenotypes that are characterized by either meiotic arrest or spermiogenic failure19.In addition to meiotic recombination, prophase I spermatocytes have an essential role in enacting complex transcriptional programs that control events during meiosis as well as the final post-meiotic differentiation stages of spermatogenesis, known as spermiogenesis22. The A-MYB protein, encoded by the Mybl1 gene, first appears in the pachytene stage of prophase I19. Once expressed, A-MYB transforms the regulatory environment by switching on a large number of enhancers that were inactive during earlier stages of spermatogenesis21. However, it remains unclear how A-MYB activates transcription during prophase I. First, we know little about how A-MYB identifies target enhancers carrying its information-poor consensus sequence, and, beyond a number of well-characterized piRNAs, the identity of its direct target genes20. Second, we do not know the mechanistic basis by which A-MYB activates transcription of its target genes. Finally, and perhaps most importantly, a general feature of transcriptional activation is extensive decondensation of chromatin. Currently, it remains unknown whether meiotic chromosome axis shortening, the formation of large chromatin loops, and the 3D genome reorganization of prophase I cells prohibit transcription and nucleosome positioning23. Therefore, a central question is how spermatocytes are able to balance the distinct, and perhaps opposing, tasks of chromatin decondensation to facilitate transcription and meiotic chromosome axis formation to aid the defining events in prophase I.Relatively little is known about how transcription programs are controlled during prophase I. The sequence-specific transcription factor A-MYB is known to activate the transcription of protein-coding genes and piRNAs by binding to proximal and distal regulatory elements and super enhancers across the genome24. Our stage-resolved maps of nascent transcription show that Pol II is loaded on chromatin and maintained in a paused state during early prophase I. Starting in pachynema, paused Pol II is released into productive elongation in a global and highly coordinated burst of transcriptional activity during which transcription is increased by ~3-fold over baseline levels. Transcriptional activation during pachynema is initiated by A-MYB, which recruits the testis-specific bromodomain protein, BRDT, to release paused Pol II into productive elongation. Both the chromatin environment and paused Pol II, which allow A-MYB binding during pachynema to activate transcription, are established by the activity of other pioneer transcription factors during earlier stages of meiosis. Finally, sites of transcriptional activity are both temporally and spatially segregated from those involved in meiotic DSB repair and recombination despite a largely shared epigenetic environment characterized by high H3K4me3 levels, allowing cells to activate transcription of essential genes and undergo DSB repair at distinct genomic loci. Taken together, these findings reveal a separation of chromatin domains, in which distinct loci are programmed during early prophase I to focus on either meiotic recombination or transcriptional activation.Here we integrated genomic data throughout meiosis to ask how spermatocytes achieve a balance between the hallmark events of prophase I and transcriptional activation. We performed a comprehensive analysis of gene expression and chromatin accessibility at discrete substages of meiotic prophase I: leptonema, zygonema, pachynema, and diplonema. To understand the mechanistic basis for the dynamic changes in transcriptional activity during meiotic recombination, we studied nascent transcription by mapping the location of transcriptionally engaged RNA polymerase II (Pol II) using length-extension chromatin run-on and sequencing (leChRO-seq) to avoid the stability-derived biases of steady-state mRNA sequencing2. Meiotic recombination occurs alongside these events, with DSBs initiated in leptonema, homolog pairing, DSB repair and the appearance of non-crossovers in zygonema, and crossovers arising in pachynema. To investigate the pattern of transcriptional activity through successive substages of prophase I, we measured the abundance of RNA polymerase II (Pol II) on chromatin by immunofluorescence on chromosome spreads using antibodies that recognize the N-terminal region of the largest Pol II subunit, RPB1 , which is associated with Pol II in a promoter-proximal paused state, and Ser2P, which marks the elongating form of Pol II26  of RPB1I26 Fig. . There w28. Transcriptional activity was nearly three times greater (2.7-fold increase) in pachynema than in leptonema/zygonema, diplonema, and round spermatids  in a more quantitative manner, we next used nuclear run-on assays to analyze each stage of prophase I and early stages during spermiogenesis. Nuclear run-ons use the activity of transcriptionally engaged Pol I-III to incorporate radioactive [α32P]CTP, which is measured using a scintillation counter. Notably, this approach has two advantages compared with immunofluorescence: first, it directly measures transcriptionally engaged RNA polymerases, and second it provides a quantitative readout of RNA polymerase abundance that is linear over many orders of magnitude29, and measure gene expression changes at the level of both transcription and steady-state mRNA abundance. To adjust for global changes in transcription observed during prophase I, we used radioactive nuclear run-on assays to normalize leChRO-seq signal. After sequencing 2–4 replicates from each molecular assay to a depth of 16–87 million uniquely mapped reads, we verified that the majority of variation in each sequencing library represented the stage during prophase I  . Staining against proteins that are defining markers of different prophase I substages (SYCP3 and γH2AX) supported a high purity in each fraction , with the majority of contamination representing elongated spermatids in all fractions. We analyzed each cell stage using three genome-wide molecular assays that measure different layers of gene regulation Fig. : ATAC-sely) Fig. .Fig. 2leAtr, Xrn1, and 1700065D16Rik all had high levels of Pol II within 60 bp of their TSS in leptonema/zygonema . Progression through pachynema and diplonema was associated with increased Pol II in the gene-body of all three genes , Local test, DESeq2; Fig. 34. Differentially transcribed genes in pachynema relative to leptonema/zygonema were enriched for gene ontologies related to fertilization, positive regulation of transcription by Pol II, and regulation of actin filament polymerization , H1fnt , and Tssk6 (involved in γH2AX formation37)  that were either up- or down-regulated across all prophase I substages  (n = 1493 genes) consisted of genes with a large increase in pachynema, which was partially, but not completely, lost during the transition between pachynema and diplonema, and sstPAC (steady-statetranscription inpachynema) (n = 766 genes) which included genes that were largely unchanged in pachynema, but had decreased signal in diplonema . Moreover, pausing indices at genes in tuPAC decreased during prophase I, whereas those in sstPAC did not change. Taken together, these results suggest genes in sstPAC are transcribed in leptonema/zygonema and pachynema and regulated in a manner that is pause-independent. Conversely, tuPAC genes transcribed in pachynema are activated by the release of Pol II from a paused state that is established in leptonema/zygonema.To identify distinct patterns of gene expression by the changes in Pol II density observed across prophase I, we clustered DE genes based on their transcription profiles in leptonema/zygonema, pachynema and diplonema according to reads falling within the TSS ± 250 bp (to capture paused Pol II), gene body, and after the polyadenylation cleavage site (PAS) Fig. . We idenema Fig. . We founema Fig. . Compareema Fig. . Genes iCcdc39 (involved in the motility of cilia and flagella), Spata1 (spermatogenesis associated protein 1), and Ybx1 and Brdt (testis-specific regulators of transcription). Genes in the sstPAC cluster were enriched for post-meiotic GO terms such as spermatid differentiation, sperm motility, and single fertilization  analysis Fig. . tuPAC gion Fig. . These rWe next asked whether the dynamic changes in transcriptional activity across prophase I alter mRNA abundance at each stage. As we do not have a way to normalize mRNA-seq data in absolute terms , we focused on examining the correlation between absolute changes in transcription and relative changes in mRNA abundance across prophase I stages. We noted reasonably strong correlations between changes at the level of transcription and mRNA abundance for DE genes -normalized RNA-seq libraries at the Coa3 (Cytochrome C Oxidase Assembly Factor 3), Cntd1 , and Becn1  loci , and the correlation between assays decreased slightly , which we clustered into 5 distinct groups based on similarities in their DNA-binding specificity , during prophase I by using dREG to identify the global initiation sites of enhancer and promoter RNAs using leChRO-seq data from each prophase I stageMyb, A-myb, and B-myb  with regulatory elements having MYB binding motifs 20. Collectively, our unbiased approach identified A-MYB as a master regulator of transcriptional changes in pachynema.The top scoring motif bound transcription factors (TFs) in the MYB family, including p < 0.0001). Genes in the sstPAC cluster were also significantly enriched for A-MYB binding compared with all genes, although the enrichment was significantly less than observed for tuPAC genes. Furthermore, A-MYB target genes were enriched for biological functions reminiscent of tuPAC genes: mRNA metabolic processing, RNA splicing, organelle localization, and histone modification . Moreover, we found that >90% of BRDT peaks coincide with A-MYB peaks, regardless of cluster, consistent with the idea that A-MYB works in part by recruiting BRDT  family protein, BRD4. BRD4 connects acetylated nucleosomes with the pause-release complex, P-TEFb, to release paused Pol II into productive elongationRDT Fig. .p-value <0.00001), consistent with a model in which A-MYB and BRDT released pre-established paused Pol II upon binding in pachynema. In fact, genes that were not bound by either A-MYB or BRDT had no change in pausing index between leptonema/zygonema and pachynema  with either JQ1 or vehicle solutions daily for 3 weeks -JQ1 (hereafter referred to as JQ1) to prevent BRDT function in prophase I cells. JQ1 inhibits the acetyl-lysine binding module of the BD1 domain of BRDT with high ligand efficiencyeks Fig. . We perfeks Fig. . We foun19, consistent with observations of both transcription and mRNA of the A-MYB-encoding gene, Mybl1 . Collectively, our results highlight A-MYB as a transcription factor which binds to poised open-chromatin regions and activates transcription by recruiting BRDT and other pause-release-associated transcriptional machinery.We asked whether A-MYB alters chromatin accessibility as its protein binding increases during entry into pachynema. A-MYB protein is reported to reach peak abundance in pachynema48, a histone methyltransferase that catalyzes the deposition of H3K4me3 and H3K36me3 near its binding sites51. PRDM9 creates a permissive chromatin environment associated with euchromatic and transcriptionally active nuclear compartments53, all of which are also histone modifications associated with active transcription.The designation and distribution of DSB and meiotic recombination hotspots is coordinated by PRDM949. As expected, DSBs were located in accessible chromatin, with a signal that peaks in leptonema/zygonema and decreases during the later stages of prophase I . We also noted no significant overlap between transcription and two other DSB markers, PRDM9 and DMC1  at single nucleotide resolution for all meiotic prophase I substages from leptonema to diplonema  during prophase I, we profiled active TREs at high resolution using a comparative analysis of nascent transcription and chromatin accessibility , or long-range contacts, are lost in pachynema2. In mice and humans, the distribution of meiotic DSBs is controlled by the binding of PRDM9 and SPO1159. To further explore the control of gene expression at DNA DSBs and recombination hotspots, we overlaid our profiles of chromatin accessibility and nascent transcription with previously published maps of PRDM9, SPO11, and DMC1 binding in C57Bl/6 males53. In particular, we found that DSB and recombination hotspots most frequently occur at regions of accessible chromatin, likely as a result of the histone methyltransferase activity of PRDM960. However, engaged Pol II was undetectable at sites of meiotic recombination in pachynema, which stands in stark contrast to the widespread transcriptional activity of meiotic chromosomes at this stage. Therefore, it is conceivable that a mechanism of active transcriptional repression exists at sites of DNA DSBs and eventual crossovers or that PRDM9, SPO11, and/or DSB repair complexes actively repel the transcriptional machinery that is activated in pachynema.The programmed formation of hundreds of DNA double-strand breaks (DSBs) is essential for initiating meiotic recombination and ensuring proper fertility62. MSUC deprives germ cells of transcripts via a megabase-scale chromatin remodeling process62. Meiotic silencing events are also associated with asynapsed regions of the X and Y chromosomes, which undergo synapsis and recombination only at the ~1 Mb pseudoautosomal region. However, unlike MSUC, meiotic sex chromosome inactivation (MSCI) is an obligatory event that arises as spermatocytes enter pachynema and is concurrent with the compartmentalization of the XY chromosomes into a heterochromatin-rich nuclear subdomain known as the sex body64. Disruption of MSCI induces mid-pachytene disruption of meiosis, leading to loss of germ cells and infertility64. It has remained unclear whether XY chromatin is permissive to Pol II recruitment and initiation at select loci. Here, we note that the sex chromosomes show a similar reduction in Pol II pausing index to that observed on the autosomes in pachynema, but without a concomitant increase in transcriptional activity. In the current study, the use of a highly sensitive assay to detect nascent RNA (leChRO-seq) allowed us to capture this pattern of pause release on the X and Y chromosomes, which would not be observable through bulk RNA-seq alone. The decrease in Pol II pausing indices on the sex chromosomes can be interpreted in several ways. First, assuming that A-MYB is capable of binding to XY chromatin in pachynema, induction of pause release on the sex chromosomes would be wholly possible, just as it would be on the autosomes. Second, mRNA expression of sex-linked loci may be low due to the formation of XY heterochromatin preventing productive elongation of Pol II or other mechanisms inducing rapid termination of paused Pol II on the sex chromosomes. Together, these data indicate that a reduction in Pol II pausing indices at XY genes in pachynema is not coupled with transcriptional activation as was observed on the autosomes. The precise nature of Pol II pause release regulation on the sex chromosomes will be a subject of investigation in our future studies.In mammalian meiosis, autosomes that fail to undergo synapsis and/or recombination are subject to a surveillance mechanism termed meiotic silencing of unsynapsed chromatin (MSUC)cis-regulatory elements and identified multiple TF pathways associated with the massive transcriptional activation observed in pachynema. We show that BRDT is recruited to highly paused genes in pachynema and is an important transcriptional co-activator of A-MYB-bound genes. Our observations of dynamic gene expression during prophase I provide insight into transcriptional regulation during key meiotic events, such as DSB initiation and repair. Indeed, these data represent an essential resource for the discovery of previously unknown regulatory elements and TFs coordinating the developmental transitions of spermatogenesis and ensuring successful male fertility.In summary, we report a comprehensive profiling of Pol II occupancy genome-wide in prophase I spermatocytes. Our study directly demonstrates the global presence of paused Pol II at the TSSs of highly expressed genes in early prophase I, thus providing a mechanism for the temporal control of transcription during spermatogenesis. We examined active The experiments described herein used mice on the C57Bl/6J or DBA/2J background, obtained from Jackson Laboratories. All mice were housed under strictly controlled conditions of temperature (range 21–24 °F), 12 h light:day cycles, and relative humidity range from 40 to 50%, with food and water ad libitum. All mouse studies were conducted with prior approval by the Cornell Institutional Animal Care and Use Committee, under protocol 2004-0063.JQ1 dosage followed the original study by Matzuk et al. (2012). Briefly, JQ1  sourced from AdooQ Bioscience  was first dissolved in DMSO at an initial concentration of 50 mg/ml and then diluted 1:10 in 10% (2-Hydroxypropyl)-β-cyclodextrin. This mixture was injected intraperitoneally (i.p.) daily into male mice at 1% of their body weight for 3 weeks for 7-week-old male mice on the DBA/2J background. Control animals received a similar i.p. daily injection of DMSO dissolved 1:10 in 10% (2-Hydroxypropyl)-β-cyclodextrin. Both control and treatment mice were weighed daily prior to injections and doses were adjusted accordingly. Three mice were used for each of the treatment and vehicle control groups.66. Testis tubules were incubated in hypertonic elution buffer  for 1 h. Small sections of tubules were minced in 100 mM sucrose solution and spread onto 1% Paraformaldehyde, 0.15% Triton X-100 coated slides. Slides were incubated in a humid chamber for 2.5 h at room temperature. Slides were dried for 30 min, washed in 0.4% Photoflo  diluted in PBS (800 ml in 200 mL PBS), 0.1% Triton X-100 diluted in PBS (1 mL and 199 mL PBS) and blocked in 10% antibody dilution buffer  diluted in PBS (20 mL and 180 mL). Primary antibodies used included rabbit-anti-SYCP3 67, mouse-anti-RNA Pol II , rat-anti-RNA Pol II Ser2P , rat-anti-RNA Pol II Ser5P , and anti-A-MYB . Primary antibodies were diluted in antibody dilution buffer, spread across the surface of the slide, and incubated at 4 °C overnight. Slides were washed for 10 min each in 0.4% Photoflo, 0.1% Triton X, and 10% antibody dilution buffer. Alexafluor™ secondary antibodies  were used for immunofluorescent staining at 37 °C for 1 h. Secondary antibodies were diluted in antibody dilution buffer and spread in a similar fashion to the primary antibodies. Slides were washed as previously described, dried, and mounted with Prolong Gold antifade (Molecular Probes). All secondary antibodies were raised specifically against Fc-fraction, Fab-fraction purified, and conjugated to Alexafluor 488 or 594.Prophase I chromosome spreads and immunofluorescent staining were prepared as previously describedImages were acquired using a Zeiss Axiophot Z1 microscope attached to a cooled charge-coupled device (CCD) Black and White Camera (Zeiss McM). The images were captured and pseudo-colored by means of ZEN 2 software . Exposure time was consistent between antibodies, cells, and mice. Brightness and contrast of images were adjusted using ImageJ  after fluorescence intensity measurements were calculated.dir=getDir("Choose the directory containing czi files");outfile=substring;outfile=outfile + ".txt";files=getFileList(dir);run;//Array of averages for each channelDave=newArray(3);fp=File.open(outfile);for  {           run;           for  {                      Stack.setPosition;                      setAutoThreshold("Otsu dark");                      run("Measure");                      Dave[i]=getResult("Mean");                      }print;           close;           }File.close(fp);A FIJI macro script was created using the available tools in FIJI. Images were in .czi file format, with DAPI in blue; SYCP3 in green; and RNA Pol II, RNA Pol II Ser2P, and RNA Pol II Ser5P in red. Intensities of DAPI, RNA Pol II, RNA Pol II Ser2P, and RNA Pol II Ser5P were determined using Otsu’s thresholding; intensities of RNA Pol II, RNA Pol II Ser2P, and RNA Pol II Ser5P were normalized to DAPI signal intensity to account for the DNA content of each cell. The script used was as follows:www.graphpad.com). Mean values were presented and alpha values were established at 0.05. All statistical analyses performed utilized One-way ANOVA with the post hoc Tukey’s multiple comparison’s test. Images of diakinesis-staged cells were not included in the statistical analysis due to low sample size.Statistical analyses of fluorescent intensity displayed in Figs. n = 40) were extracted and decapsulated prior to enrichment of prophase I staged cell types using the gravitational cell separation method, STAPUT, which is based on separation by cell diameter and density at unit gravity68. Purity of collected fractions was determined by staining against proteins defining prophase I substages, specifically SYCP3 and γH2AX (EMD Millipore 05-636), and imaged on a Zeiss Axiophot with Zen 2.0 software. Leptotene and zygotene, pachytene, and diplotene cells were at approximately 87%, 84%, and 81% purity, respectively, with potential contamination from spermatogenic cells of either earlier or later developmental timing. Round and elongating spermatids were identified by DAPI staining and collected at l00% enrichment. Cells isolated by STAPUT were used as input for leChRO-seq (n = 4) and RNA-seq (n = 3) library preparation.Testes from adult wild-type mice  were extracted and decapsulated prior to enrichment of prophase I staged cell types using Flow Cytometric Analysis and Fluorescence Activated Cell Sorting (FACS)69. Prior to sorting, testicular single cells were stained with Hoechst dye and Propidium Iodide for selection based on DNA content and dead cell exclusion, respectively. Data analysis was done using the BD FACSDiva Software v6.1.3 on a FACSAria II cell sorter. Hoescht was excited using the 355 nm laser. Sorting flow rate was adjusted to 7–32 events/second. Supplementary Fig. n = 2).Testes from adult wild-type mice , 1x Protease Inhibitor Cocktail . Samples were vigorously vortexed for 1 min. 500 ml of NUN buffer was added to each sample and vortexed for another 30 s. The samples were then incubated on ice for 30 min with a brief vortex every 10 min. Chromatin samples were centrifuged at 12,500 × g for 30 min at 4 °C. Following removal of the NUN buffer from the chromatin pellet, the chromatin pellet was washed with 1 ml 50 mM Tris-HCl, pH 7.5, with 40 units per ml RNase inhibitor, centrifuged at 10,000 × g for 5 min at 4 °C, at which point the buffer was discarded for a total of three washes. After washing, 100 ml of chromatin storage buffer 2), 0.1 mM EDTA, 5 mM DTT, 40 units per ml RNase inhibitor) was added to each sample. The samples were sonicated with a Bioruptor on high power, with a cycle time of 10 min with cycle durations of 30 s on and 30 s off. The sonication was repeated up to four times as needed to get the chromatin pellet into suspension. The samples were stored at −80 °C.The methods of chromatin isolation were based on work described by Chu et al., 2018n = 17) or round and elongated spermatids (n = 4). Prior to the nuclear run-on assay, DNA content of isolated chromatin was measured to ensure equal input of material per sample (500 ng of DNA). Three 10-fold serial dilutions of chromatin isolated from two Jurkat samples  was also used to standardize each nuclear run-on experiment to account for radioactive decay of [α32P]CTP. The nuclear run-on was started by mixing isolated chromatin with 50 ml 2x chromatin run-on buffer , 400 µM GTP (NEB N0450S), 400 µM UTP (NEB N0450S), 40 mM 32P-CTP (Perkin Elmer BLU008H), 0.8 units/µl SUPERase in RNase Inhibitor (Life Technologies AM2694), and 1% Sarkosyl (Fischer Scientific #AC612075000)). The run-on reaction was incubated at 37 °C for 10 min and then stopped by adding Trizol LS (Life Technologies 10296-101). RNA was extracted with 1-bromo-3-chloropropane, pelleted with GlycoBlue treated water, and washed with 100% and 75% ethanol. The RNA was resuspended in diethyl pyrocarbonate (DEPC) H2O and any free nucleotides were captured by the use of two P-30 columns. All samples were assessed for radioactive levels on an LS Beta Counter. The relative transcriptional activity of leptonema/zygonema (LZ), pachynema (P), and diplonema (D) was calculated using the formula below:Calculate line of best fit for observed CPM of Jurkat standards: y = mx + b, where y = rate of ionization events per minute (CPM); and x = DNA concentrationCalculate expected CPM for samples using line of best fit: substitute x with measured DNA concentration of sampleRelative transcriptional activity:LZ, P, and D represent the relative transcriptional activity of each stage, and were solved for using a system of linear equationsChromatin was isolated from STAPUT-sorted fractions containing either mixed populations of prophase I cells  of the relative transcriptional activity for each cell type were presented in Fig. The relative transcriptional activity of round and elongated spermatids, enriched at 100%, was calculated from the 24. Briefly, chromatin from enriched prophase I substages was mixed with 100 µl 2x chromatin run-on buffer , 40 µM Biotin-11-CTP (Perkin Elmer NEL542001EA), 400 µM GTP (NEB N0450S), 40 µM Biotin-11-UTP (Perkin Elmer NEL543001EA), 0.8 units/µl SUPERase in RNase Inhibitor (Life Technologies AM2694), and 1% Sarkosyl (ThermoFisher #AC612075000)). The run-on reaction was incubated at 37 ˚C for 5 min and then stopped by adding Trizol LS (ThermoFisher 10296010). RNA was extracted from Trizol/chloroform mixture and was then pelleted with GlycoBlue and ethanol precipitation. Nascent RNA was purified by binding streptavidin beads (NEB S1421S) and washed as described by Mahat et al., 201670. The RNA was removed from beads by Trizol (ThermoFisher # 15596026) and then followed with the 3′ adapter ligation with T4 RNA Ligase I (NEB M0204L). After this, a second bead binding was performed followed by a 5′ de-capping using RppH (NEB M0356S). The 5′ end of the transcript was phosphorylated using PNK (NEB M0201L) and then purified with Trizol. After this, a 5′ adapter was ligated onto the RNA transcript, and a third bead binding was performed by reverse transcription reaction in order to generate cDNA (ThermoFisher # 18090010). The cDNA was amplified (NEB M0491L) in order to generate the leChRO-seq libraries which were prepared using manufacturer’s protocol (Illumina) and paired-end sequenced by means of Illumina NextSeq500 at Cornell University Biotechnology Resource Center.leChRO-seq libraries were prepared as previously described Chu et al., 2018https://github.com/Danko-Lab/utils/tree/master/proseq). The libraries were prepared by means of adapters containing a specific molecular identifier. For these specific transcripts, PCR duplicates were removed using PRINSEQ lite71. The adapters were cut from the 3′ end of remaining reads using cutadapt with a 10% error rate72. Reads from this data were mapped using BWA73 to the mouse reference genome (mm10) in addition to a single copy of the Pol I ribosomal RNA transcription unit (GenBank ID #U13369.1). The specific location of the RNA polymerase active site was described by means of a single base which denoted the 5′ end of the nascent RNA. This information corresponded to the position of the 3′ end of each sequenced read. Mapped reads were converted to bigWig format using BedTools74. Libraries were normalized using the following formula:LZ and P represent the relative transcriptional activity of each stage, and (library size) P ÷ LZ) represents the library normalization factors.In order to align leChRO-seq data, a publically available script was used . Total RNA was purified using TrizolLS according to the commercial protocol with the following additions: after the first phase separation, an additional chloroform extraction step of the aqueous layer was performed using Phase-lock Gel heavy tubes (Quanta Biosciences); addition of 1ul Glyco-blue (Thermo Fisher) immediately prior to isopropanol precipitation; two washes of the RNA pellet with 75% ethanol. If the RNA integrity results indicated co-purified genomic DNA, it was removed with the RapidOUT DNA Removal kit (Thermo Fisher). RNA sample quality was confirmed by spectrophotometry (Nanodrop) to determine concentration and chemical purity (A260/230 and A260/280 ratios) and with a Fragment Analyzer  to determine RNA integrity. PolyA+ RNA was isolated with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). TruSeq-barcoded RNAseq libraries were generated with the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs). Each library was quantified with a Qubit 2.0  and the size distribution was determined with a Fragment Analyzer  prior to pooling. Libraries were sequenced on a NextSeq500 instrument (Illumina). At least 20 M single-end 75 bp reads were generated per library.76. Mapped reads were converted to bigWig format using BedTools. To normalize for sequencing depth, libraries were scaled using reads per kilobase per million mapped reads (RPKM). The WashU Epigenome Browser was used for visualization of RNA-seq libraries.Reads were trimmed for low quality and adaptor sequences with cutadapt v1.8; parameters: -m 50 -q 20 -a AGATCGGAAGAGCACACGTCTGAACTCCAG–match-read-wildcards. Reads were mapped to the reference genome (mm10.rRNA.fa.gz) using STAR v2.5.3a78.Prepare nuclei: In order to prepare nuclei, 50,000 cells isolated by FACS were spun at 500 × g for 5 min immediately after sorting, followed by a wash using 50 µl of cold 1 x PBS and centrifugation at 500 × g for 5 min. Cells were then lysed using a cold lysis buffer . Immediately following lysis, nuclei were spun at 500 × g for 10 min in a refrigerated centrifuge. In order to avoid cell loss during nuclei preparation, a fixed-angle centrifuge was used and cells were carefully pipetted away from the pellet after centrifugation. Nuclei were stored at −80 °C.Transpose and purify. Stored nuclei were thawed on ice and the pellet was resuspended in a transposase reaction mixture , and 22.5 µl nuclease-free water). The transposition reaction mixture was carried out for 30 min at 37 °C. Immediately following transposition, the sample was purified by means of a Qiagen MinElute kit.PCR. After purification, the library of fragments was amplified using 1 x NEBnext PCR master mix and 1.25 µM of custom Nextera PCR primers 1 and 2. The following PCR conditions were utilized: 72 °C for 5 min; 98 °C for 30 s; and thermocycling at 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min. In order to reduce GC and size bias in the PCR analysis, PCR reactions were monitored using qPCR in order to stop amplification prior to saturation. This was done by amplification of the full libraries for five cycles, after which an aliquot of the PCR reaction was taken and 10 µl of the PCR cocktail containing Sybr Green at a final concentration of 0.6x was added. This reaction was run for 20 cycles in order to determine the additional necessary number of cycles for the remaining 45 µl reaction. The libraries were purified by means of a Qiagen PCR cleanup kit that yielded a final library concentration of ~30 nM in 20 µl. These libraries were amplified for a total of 10–12 cycles in order to generate the ATAC-seq libraries, which were prepared using the manufacturer’s protocol (Illumina) and paired-end sequenced by means of Illumina NextSeq500 at Cornell University Biotechnology Resource Center.This methodology is directly based on the original design79 and filtered for uniquely mapping pairs with a custom python script80. Duplicate and multiple aligning reads were removed from the analysis with picardtools v2.1.1 “MarkDuplicates.” Mapped reads were converted to bigWig format using BedTools. To normalize for sequencing depth, libraries were scaled using reads per kilobase sequence per million mapped reads (RPM). ATAC-seq peaks were called on replicate samples using Genrich v0.6 . All peaks called by the peak calling software were included in our analysis. Mean FRiP scores for ATAC-seq libraries was 56.7%. The WashU Epigenome Browser was used for visualization of ATAC-seq libraries.ATAC-seq sequencing reads were aligned to the mouse reference genome (mm10) using Bowtie281 using the prcomp function of the R stats package81. Sequencing reads from each genomics assay were counted in gene bodies of all protein-coding genes that were shown to be expressed from the leChRO-seq data and used as input for the PCA analysis. We compared the first two PCs for all datasets.Principle component analysis of the leChRO-seq, RNA-seq, and ATAC-seq datasets were calculated in R v3.4.2bigWig package82 in R v3.4.2 in the intervals between (1) the annotated transcription start site (TSS) to 250 bp downstream of the annotated TSS; (2) 250 bp downstream of the annotated TSS to the 3′ end of the gene; (3) the 3′ end of the gene to 5000 bp downstream of the 3′ end of the gene. These windows were selected to count reads within the pause peak, gene body, and post poly(A) signals of all annotated genes. We limited analyses to gene annotations longer than 500 bp in length and gene annotations in which the end of the gene was at least 5000 bp away from the TSS of neighboring genes. Differential expression analysis was conducted using DESeq234 v1.32.0  and differentially expressed genes were defined as those with a false discovery rate (FDR) less than 0.05. Normalization of the read count matrix was achieved by manually selecting DESeq2 size factors by dividing the read count matrix by library size and multiplying by the relative transcriptional activity of the prophase I substages for each replicate (above). DESeq2 was conducted to compare differential expression between prophase I substages.We counted reads using the https://github.com/Danko-Lab/histone-mark-imputation/figures/ScaledMetaPlotFunctions.R). This script plots the average leChRO-seq signal intensity for all annotated genes in Fig. We used a publicly available script to generate leChRO metaplots  on the normalized read count matrix for all gene intervals of differentially expressed genes (above). The read count matrix for P and D were normalized to the read count for LZ. The pheatmap package84 in R v4.0.5 was used to generate the heatmap in Fig. The Python package 29. A complete description of dREG can be found here: https://github.com/Danko-Lab/dREG.dREG was run using the default settings to identify putative transcriptional regulatory elements (TREs) from leChRO-seq librariesbigWig package in R v3.4.2. Differential expression of dREG peaks comparing LZ to P and P to D was performed using DESeq2 . Volcano plots reflecting DE of dREG peaks were made using the EnhancedVolcano package85 v1.10.0 in R. dREG peaks with a log2FC > 1 between LZ and P were selected to identify the top DE dREG peaks  for transcription factor (TF) binding motif enrichment. HOMER40 was used to identify enriched TF binding motifs within upregulated dREG peaks between LZ and P, allowing multiple motifs per peak and searching for up to 25 motifs 100 bp upstream and downstream from the center of each peak. Donut plot of enriched TF families was generated using the lessR package86 v4.0.5 in R. MotifScan was used to determine the genomic position of upregulated TREs in P containing the A-MYB binding motif87. BedTools “closest” was used to identify putative DE target genes with an FDR < 0.05 for each TRE containing the A-MYB binding motif. Putative DE target genes were defined as those nearest to each TRE containing the A-MYB motif within 50 kb.Briefly, we merged dREG peaks from all prophase I stages using BedTools “merge” and counted leChRO-seq reads for each prophase I substage using the clusterProfiler38 v4.0.5. In Fig. a = 0.05) was applied to control for FDR. GO annotation enrichment for biological processes found in Fig. dotplot function of the Enrichplot88 v1.12.2 package. Dot plots were ranked by adjusted p-value and gene set size. GO annotation enrichment for biological processes found in Fig. cnetplot function of the Enrichplot package.The GO enrichment analysis shown in Figs. 20 (GSE44588) and BRDT ChIP-seq peaks44 (GSE98489) were remapped from the mm9 to mm10 reference genome and mm8 to mm10 reference genome, respectively, using the UCSC LiftOver tool. Bar plots of the percent of A-MYB or BRDT peaks overlapping with the TSSs of genes in the tuPAC cluster, sstPAC cluster, or all transcription units (TUs)  Fig.  were genUs) Fig.  from leC90 v1.5.2 to the mm10 genome and tximport91 v1.20.0 was used to summarize transcript-level to gene-level abundance estimates in R v4.0.5. Raw sequencing counts from Salmon were DESeq2 normalized (parameters: rowSums(counts(dds)) > 1). DE genes were defined as those with an FDR less than 0.05. Scatterplots comparing the log2 fold change and log FPKM of DE genes or all genes using leChRO-seq and RNA-seq data as input presented in Supplementary Fig. ggplot292.Quantification of sequencing reads was performed using Salmon93 and annotated TSSs using BedTools intersect. Counts of reads falling into merged peak files were determined using the BedTools multicov function for each sample, and were subsequently RPKM normalized. Mean values ± 95% confidence intervals of RPKM for each class of ATAC-seq peaks were presented in Fig. 93.Peak calls from biological replicates for each prophase I substage were used to determine total unique peak sets for each substage. Peak calls representing all of prophase I were then combined and merged using BedTools. Both transcribed and non-transcribed ATAC-seq peaks were then compared to a publicly available H3K4me3 dataset40 was used to identify enriched TF binding motifs in ATAC-seq peaks, allowing multiple motifs per peak and searching for up to 25 motifs 100 bp upstream and downstream from the center of each peak. MotifScan was used to determine the genomic position of accessible regions containing the A-MYB, RFX2, and CREB binding motifs87.HOMERGSE84689), and DMC1 ChIP-seq peaks (GSE35498)) are enriched in accessible chromatin, based on overlap with ATAC-seq peaks at the different stages of meiotic prophase I53. Using the center of the dREG or the DSB marker peaks, we asked what percentage of the ATAC-seq peaks overlap with either of these markers by counting the number of ATAC-seq peaks with values greater than zero for any of the markers following BedTools “intersect” with -C function, at all three stages. To ask if the calculated overlaps between ATAC-seq peaks and the different markers are different than what is expected by chance, we randomly shuffled the positions of ATAC-seq peaks across the genome, while preserving their size, preventing overlaps or changes in chromosome distribution with BedTools “shuffle -chrom -noOverlapping”, 1000 times. In each of these 1000 iterations, we asked what percentage of the shuffled ATAC-seq peaks overlapped with the dREG and DSB markers. We then calculated an empirical p-value for each marker by taking the fraction of the iterations that resulted in ATAC-seq peak overlaps greater than the observed overlap.We first asked whether transcription initiation, marked by dREG peaks, and the DSB markers  and the DSB markers was smaller than the observed co-occurrence calculated.We then asked if the accessible genomic regions presenting transcription initiation occur near or are separated from sites of DSBs. We considered a window of 5 kb around the center of ATAC-seq peaks in each of the stages and asked what fraction of these expanded peaks overlapped with each of the markers, including dREG and DSB markers. We then calculated the co-occurrence proportion of dREG and DSB markers in the same expanded ATAC-seq peak. We defined the column of dREG overlapping expanded ATAC-seq peaks as “transcribed ATAC peaks” and randomly shuffled this column 1000 times while reassessing the co-occurrence in each iteration. The empirical Figures No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Arabidopsis thaliana, has hardly been investigated, except for a few early studies that addressed the role of a TATA box and substitutions in it in plant transcription systems. This is despite the fact that the interaction of TBP with TATA boxes and their variants can be used to regulate transcription. In this review, we examine the roles of some general transcription factors in the assembly of the basal transcription complex, as well as functions of TATA boxes of the model plant A. thaliana. We review examples showing not only the involvement of TATA boxes in the initiation of transcription machinery assembly but also their indirect participation in plant adaptation to environmental conditions in responses to light and other phenomena. Examples of an influence of the expression levels of A. thaliana TBP1 and TBP2 on morphological traits of the plants are also examined. We summarize available functional data on these two early players that trigger the assembly of transcription machinery. This information will deepen the understanding of the mechanisms underlying transcription by Pol II in plants and will help to utilize the functions of the interaction of TBP with TATA boxes in practice.For transcription initiation by RNA polymerase II (Pol II), all eukaryotes require assembly of basal transcription machinery on the core promoter, a region located approximately in the locus spanning a transcription start site . Although Pol II is a complex multi-subunit enzyme conserved among all eukaryotes, it cannot initiate transcription without the participation of many other proteins. Transcription initiation on TATA-containing promoters requires the assembly of the preinitiation complex; this process is triggered by an interaction of TATA-binding protein ) with a TATA box. The interaction of TBP with various TATA boxes in plants, in particular  Development of multicellular organisms and the uniqueness of each cell are implemented through the expression of certain genes at the right time in the right place. This phenomenon requires a complicated modulation of gene expression by regulatory factors interacting with specific DNA sequences and among themselves; these processes are critical for the growth, development, and survival of organisms. The foundation of this phenomenon is the fact that, while the number of protein-coding genes has remained fairly constant throughout the evolution of metazoans, the number of regulatory DNA elements has increased dramatically . The modIn eukaryotes, transcription is carried out by three DNA-dependent RNA polymerases. Pol I and Pol II produce ribosomal RNAs and messenger RNAs for ribosome assembly and protein synthesis, respectively. Pol III specializes in synthesizing 5S rRNA, transfer RNAs (tRNAs), U6 spliceosomal RNA (U6 snRNA), and small noncoding RNAs, including 7SL RNA, 7SK RNA, and RNase P RNA . It shouDrosophila development-related genes [Bridge is a rare element of a core promoter; it contains the first and third subregions MTE and DPE and is the site of interaction of TAF6 and TAF9 subunits of TFIID (transcription factor II D) [Drosophila to humans and their functions depend on the presence of a functional Inr; they are enriched in TATA-free promoters [The core promoter is the primary site for the assembly of the preinitiation complex ,8. It coed genes ,9,14. Bror II D) ,16. TFIIor II D) . The MTEromoters . Figure romoters ,19.u and BREd, respectively) [Recognition of the core promoter by transcription machinery is necessary for proper positioning and assembly of the preinitiation complex. Biochemical studies have shown that TBP binds to a TATA element in both directions . It follctively) ,22, whicctively)  and for ctively)  and the ctively) . Crystalctively) . Aside fctively) . The cryctively) . By crysctively) . Thus, tThe architecture and composition of the core promoter determine the initiation of transcription—starting with the assembly of the preinitiation complex specific for this type of promoter—and are responsible for the implementation of enhancer–promoter specificity , thereby predetermining the course of the subsequent stages of transcription, such as the promoter release, Pol II arrest, or the transition to productive elongation. The core promoter also participates in the regulation of termination, polyadenylation, RNA polymerase recycling, and the modulation of translation ,28. DiffTBP is a subunit of a general  transcription factor called TFIID. It is known that the most important function of TBP in TBP- and TFIID-containing preinitiation complexes is promoter DNA bending  Figure , which mObviously, on TATA-containing promoters, the DNA–protein interaction plays a decisive role, whereas on TATA-free promoters, the specificity of the interaction of TBP is implemented via mechanisms of direct and indirect interaction with activator proteins and TBP-associated factors. On a TATA-free but TFIID-dependent promoter, the positioning of TBP near nucleotide position −30 is mediated by the interaction of TBP-associated factors with other core promoter elements, by the interaction of TFIIB with BRE, and by protein–protein interactions of TBP and of TBP-associated factors with activators ,17,34.Until recently, it has been believed that TBP is a general transcription factor necessary for the transcription of all genes. Today, three paralogs of TBP are already known; these are so-called TBP-related factors: TRF1, TRF2, and TRF3. All of them share homology with the core domain of TBP. TRF1 and TRF3 can interact with a TATA box, but TRF2 cannot because it has lost the phenylalanine residue that intercalates into DNA . The corDrosophila, of yeast, and of Arabidopsis has shown that it consists of a phylogenetically conserved carboxyterminal domain 180 amino acid residues long and a variable amino-terminal domain [Analysis of the amino acid sequence of TBP of humans, of l domain . The C-tl domain ,38 and cl domain ; therefol domain ,41.Arabidopsis thaliana has revealed that the C-terminal domain of eukaryotic TBP has a pseudo-symmetrical structure formed by ten β-folds  and by four α-helices  [Crystallographic analysis of full-length TBP of olecule) .AdML promoter carrying a TATA box in a complex with yeast or human TBP has been demonstrated in original experiments involving an electrophoretic gel shift assay [S. cerevisiae TBP with the TATA box of the CYC1 promoter [A. thaliana with the TATA box of the AdML promoter [The bending of the ft assay . In 1993promoter  and of fpromoter . It was promoter  between promoter ,44. The promoter .Unlike most DNA-binding proteins, which specifically interact with the major groove of DNA to form hydrogen bonds between amino acid residues and nucleotides, TBP when choosing a target interacts with the minor groove of DNA and recognizes structural features such as flexibility or deformability . The speDrosophila histone genes) and is best known is the TATA box [Drosophila [A. thaliana [The eukaryotic core promoter element that was identified first , whereathaliana . Studiesthaliana . After athaliana  construcIn addition to the table format, frequency matrices are presented as a picture: a sequence logo . Each coIn the Bucher frequency matrix, T at the third position has the greatest weight (frequency of occurrence); next, As at the second and fourth positions have the same weight; As at the fifth and sixth position have less weight.In contrast to the frequency matrix of mammals, in the frequency matrix of plants, the greatest weight belongs to A at the fourth position . SomewhaThe literature data indicate wide variations of the TATA box sequence among natural promoters. Different researchers give dissimilar estimates of the prevalence of TATA boxes. For example, it is reported in Ref.  that ~24S. cerevisiae and demonstrated that it is the general transcription factor that is recruited to most promoters of genes transcribed by Pol II, where SAGA plays a critical role in mRNA synthesis regardless of the promoter type . In this regard, SAGA can be compared to the mediator, which is required for the whole transcription by Pol II and stimulates the formation of the preinitiation complex. The authors of Ref. [It has been shown in yeast that expression regulation fundamentally differs between genes containing the TATA consensus and genes containing a TATA-like element. Genome-wide studies indicate that transcription of approximately 10% of the yeast genome is predominantly regulated by SAGA and 90% of the genome by TFIID. Most SAGA-dependent yeast genes are stress-induced and tightly regulated, whereas TFIID-dependent yeast genes are mostly housekeeping genes . SAGA-de of Ref.  concludeAlthough the TATA box (and TATA-like elements) is usually only one of several synergistically acting core promoter elements ensuring precise positioning of TBP on a promoter, TATA-containing promoters can be highly sensitive to substitutions in their sequence. It is known that, for different promoters, identical substitutions in the TATA box have dissimilar effects on promoter activity ,68,69.E4 (TATATATA), or asymmetrical, as in AdML (TATAAAAG) [The interaction of TBP with a TATA-like element largely depends on flanking sequences. Flanking-sequence motifs affecting the TBP–TATA interaction are unique to certain TATA boxes . It is rATAAAAG) .A high information content may point to the presence of functional binding sites for other transcription factors, e.g., BRE, in the flanks of a TATA box .Results implying an effect of flanking sequences on the affinity of oligonucleotides for TBP are presented in Ref. . ExperimIt has been traditionally believed that, on TATA-containing promoters, the assembly of the preinitiation complex is triggered by the interaction of TBP, which is a component of the general transcription factor TFIID, with the TATA box. This sequential assembly model was described in 1994  and can AdML promoter containing the consensus TATA box (sequence TATAAAAG), the DCE (downstream core element) [Although the TATA box is regarded as an element capable of directing transcription independently of an Inr, experimental evidence  suggestselement) , and theAnother pathway of preinitiation complex assembly is assumed to be mediated by the recruitment of a preassembled holoenzyme complex of Pol II, which comprises Pol II, general transcription factors , and proteins partaking in other cellular functions .The large number of proteins required for transcription has always raised the question of spatial organization of transcription in the nucleus and how quickly the factors required at a given moment can be delivered. Ultra-high-resolution microscopy of live cells has revealed the dynamic foci of Pol II, which are termed concentrates or condensates ,77,78,79Mus musculus and Homo sapiens), the GC content (%) exceeds the AT content in all regions except for the region from −30 to −26 bp, where these percentages are almost equal. In contrast, A. thaliana core promoters contain the highest AT content in all regions, peaking between positions −35 and −26 bp [Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio contain approximately equal AT and GC percentages, whereas the region −31 to −26 bp is also enriched with nucleotides A and T [D. melanogaster, D. rerio, and of C. elegans, the occurrence of TATA reaches a maximum at position −28 or −29 bp, whereas in A. thaliana, this maximum is at −31 bp relative to a transcription start site. The maximum of occurrence of AAAA is at position −26 bp in mammals, D. melanogaster, D. rerio, and C. elegans and at −28 bp in A. thaliana. The distribution of dinucleotides and tetranucleotides in the region of a transcription start site varies fairly widely among species. Position +1 is mostly occupied by A in almost all species [Research into the structural characteristics of DNA shows that, for promoters of mammals , have documented a statistically significant difference  in the prevalence of each element, except for the TATA box, DPE, and Y-patches. Nucleosome-free DNA in the region of core promoters of six studied species of multicellular eukaryotes has properties that are necessary for structural changes that promote the binding of TBP and the formation of an open promoter complex [er genes , which her genes , upon wher genes . Unlike er genes ,90; ~18% islands ,91,92. T islands ,93,94. G islands . It shou of Ref. , which i complex . These pA. thaliana, compared with genes of yeast, Drosophila, humans, and other mammals. For example, Mukumoto et al. [A. thaliana TATA boxes on in vitro transcription in a HeLa nuclear extract, where human TBP is replaced by TBP from A. thaliana , which are effective in the transcription of eukaryotic genes, were also effective in transcription with either A. thaliana or human TBP. In that study, substitutions from A or T to C or G at position 2, 3, 4, or 5 strongly reduced transcriptional efficiency, whereas substitutions at position 1, 6, or 7 had a weaker impact. Among the mutant TATA elements, sequences TAGAGATA and GAGAGAGA resulted in the complete suppression of transcription.There are very few experimental studies on the interaction of TBP with TATA elements in promoters of plant genes, in particular in o et al.  have invthaliana . Those aCaMVsynT-3 gene in A. thaliana protoplasts and found that, if this sequence is replaced with either TAcgAATA or TATAcgTA, the gene expression is inhibited by 95%   and fifth positions (TATA boxes No. 15 and 16) and A at the fourth position (TATA boxes No. 12 and 13) and at the sixth position (TATA boxes No. 17–19), in good agreement with the Bucher and Shahmuradov frequency matrices [For TATA box No. 4 in the tobacco transcription system, the activity is 20% of that of the consensus sequence TATATATA; in the HeLa system it is 40%; in ference) . For TATt matrix . Accordimatrices ,58. Amonmatrices ; furtherHow the interactions of TBP with TATA boxes differ and how much these interactions correlate with transcriptional efficiency remains unknown, although this information may be important for the design of superpromoters.A. thaliana seedling development suggest that light modulates many pathways of plant growth and development [1C2A3C4T5A6T7A8T9A10T11A12G13) [7 or A8 with G/C has almost no effect on expression in the dark, regardless of whether the light-sensitive element Ire is present   on light present . In cont9 substitutions on the expression of the minimal Pmec promoter and the same TATA box prototype sequence was cloned into the pBI101 binary vector containing the gusA reporter gene. Tobacco leaves were transformed with the resulting constructs. The impact on transcription was evaluated in the presence of various sequences of an activator located upstream of the transcription start site. In contrast to mutations at the seventh and eighth positions [9 by eight–nine-fold and C9 by three–four-fold compared with T9. In the dark, the expression was greater approximately five- and three-fold, respectively. Experiments with nuclear proteins from seedlings grown in the dark or under light have shown that sequence variations within a TATA box regulate the assembly of alternative transcription complexes . The samositions , mutatioPmec lines with the G9 mutation in the TATA box manifested more than 22-fold stronger expression compared with the transgenic Pmec lines having the TATA box prototype. From their results, those authors [9 to G or C in the TATA box gives more favorable sequence architecture, which enhances transcription. The same authors [9 and C9 give rise to multiple transcription start sites, in contrast to the construct containing the prototype A1C2A3C4T5A6T7A8T9A10T11A12G13, with which only one transcription start site was observed [PAL promoter.Without the activator, the transgenic  authors  conclude authors  also revobserved . From thobserved , a reasoobserved  also indobserved  that theA. thaliana, the authors of Ref. [Arabidopsis to an attack by various insect species (caterpillars and aphids). Insects caused various changes in the expression of genes associated with amino acid metabolism. In response to the caterpillar attack, there was activation of defense gene coding for proteins involved in biosynthesis as well as activation of glucosinolate biosynthesis. Of note, the biochemical reactions seemed to be specific to herbivorous insect species; some insects suppressed or did not induce defense responses in the plants. It was shown in that report that the set of caterpillar-activated genes is enriched with the TATA boxes that may play a part in the regulation of the biotic-stress response . Genes devoid of TATA boxes perform housekeeping functions and probably do not require much transcriptional regulation.With the help of genome-wide microarrays of  of Ref.  have exaArabidopsis, maize, and sorghum. They found that core promoters containing a TATA box are four times stronger than promoters without a TATA box, especially when a TATA box is located in the region 23 to 59 bp upstream of a transcription start site, where most TATA boxes are situated in the promoters of Arabidopsis, maize, and sorghum. The substitution of one or two T nucleotides with G in the main TATA motif lowered transcriptional activity. Similarly, promoter strength increased when the canonical TATA box was inserted into a promoter devoid of TATA. The mutated version of the TATA box did not have this effect. To determine which promoter characteristics affect sensitivity to an enhancer, those authors analyzed the elements that influence promoter strength and found that promoters with a TATA box were 67% more sensitive to the enhancer nucleotide sequence than promoters without a TATA box.Some researchers  have conArabidopsis plants with a knockout of the IRT1 gene (iron-regulatory transporter 1) encoding a transporter of Fe  suffer from chlorosis and die. By modifying the promoter of the IRT1 transporter gene, namely by inserting an additional TATA box, other investigators [Malus) adapt to a deficit of iron ions . This modification raised the gene expression and Fe uptake. To further characterize the influence of the TATA box insertion on the activity of the IRT1 promoter, the authors of Ref. [Arabidopsis protoplasts via the addition of one to three TATA boxes fused to a GUS reporter. Gene expression was measured relative to GFP (green fluorescent protein) expression driven by the constitutive 35S promoter. The results uncovered a clear-cut positive correlation between the number of TATA box inserts and promoter activity. The binding of transcription factor TFIID to the IRT1 promoter was enhanced by the presence of the TATA box insert, yielding a greater amount of IRT1 transcripts and a higher Fe uptake. Those authors [As a consequence of the development of the Earth’s aerobic atmosphere, the need for iron in plants has risen and its deficit has become a limiting factor for plant growth. It is reported in Ref.  that Aratigators  have sho of Ref.  assayed  authors  believe A. thaliana) contain a TATA box in contrast to genes related to protein folding, where only 20% contain a TATA box. Those authors theorize that the presence of a TATA box in a promoter creates a conformation for a transcription factor that promotes precise and rapid transcription initiation [Different categories of genes (according to their participation in cellular processes) have dissimilar frequencies of TATA boxes ; for exaitiation . This isitiation  that proitiation ,111, e.gThus, we can say the following about the main functions of the TATA box: the overall magnitude of expression of TATA-containing genes correlates well with the sequence of the TATA box  ,113; mosArabidopsis is known to have two genes encoding TBP [Arabidopsis, these isoforms differ by 10% and may have major differences in protein-binding surfaces [A. thaliana [Arabidopsis. In TBP1, position 193 contains arginine in Arabidopsis, alanine in maize, and serine in both TBP2s. Position 198 features valine in both TBP1s and isoleucine in both TBP2s. This divergence may lead to isoform-specific functional differences in interactions with other proteins [ding TBP ,118,119;ding TBP  and potading TBP . In Arabsurfaces . The exathaliana  by X-rayproteins .Arabidopsis [Drosophila [A. thaliana TBP2) contacts TFIIB the most by forming a firm salt bridge and two hydrogen bonds. All 12 residues in the conserved core of human TFIIB that bind to TBP are conserved in Drosophila, 10 are conserved in A. thaliana, and 8 in yeast [Gal4 promoter versus more complex natural promoters); furthermore, the two stirrup glutamine residues of TBP differ in importance, with E146 being more crucial than E144 [A comparison of protein sequences suggests that, among eukaryotic organisms, the TBP–TFIIB interaction is highly conserved, involving eight amino acid residues of TBP and 12 of TFIIB . Four ofbidopsis , humans bidopsis , Drosophosophila , and yeaosophila . Of thesin yeast . The depin yeast . In HeLain yeast  and in phan E144 .A. thaliana, indicating that an elevated concentration of this protein can impair cell differentiation in the leaf embryo or in young shoots, thereby turning them into meristem-like structures [TBP2 in transgenic A. thaliana plants induces proliferation of apical shoots and the expression of some genes (STM and KNAT1) regulating the shoot meristem undergoes changes. All these findings suggest that the expression level of the TBP gene may play a major part in the control of shoot formation and plant morphology [The upregulation of TBP2 has been detected in apical shoots of ructures . Overexprphology . A. thaliana), with mediator subunits, with Pol II, and with central-cell-specific AGAMOUS-like transcription factors. Additionally, CCG interacts with TBP1 and Pol II as a TFIIB-like transcription factor. CBP1 is expressed in many vegetative tissues, especially in the central cell during reproductive growth. Some scientists [In flowering plants, spermatozoa are delivered to the embryo sac by a pollen tube guided by female signals. Both gametic and synergid cells contribute to the guiding of pollen tubes. Synergids secrete peptide signals that anchor the tube, whereas the function of gametic cells is unknown. Some investigators  have ideientists  believe Arabidopsis (TBP1 and TBP2) by means of three types of promoters: from the U2 gene of small nuclear RNA and from mRNA genes read by Pol II as well as from the U6 gene of small nuclear RNA generated by RNA polymerase III. Transcriptional conditions for a yeast system [U2 gene was four-fold higher with the mutant TATA box TGTAAA and two-fold higher with mutant TATA boxes, in which TATAAA was replaced with TCTAAA, TAGAAA, or TATAGA. Mutant human TBP also worked with TGTAAA (a mutant sequence) in all three gene types [Arabidopsis isoforms of TBP are functionally identical if we compare transcriptional systems of Pol II and III in plant cells.The authors of Ref.  have doct system  were empt system . Expressne types . TherefoArabidopsis, as in other eukaryotes, TBP and TBP-associated factor (TAF)-like proteins have been identified [A. thaliana TFIID [Arabidopsis possesses two isoforms of TAF1. One isoform of TAF1 has a domain structure homologous to that of other eukaryotes, while the second isoform differs from the first one in that its N-terminal domain is shorter by 91 amino acid residues [In entified ,133,134.na TFIID  has showresidues .Arabidopsis isoforms of TBP interact only with the N terminus of TAF1, the portion of the protein predicted to contain the N-terminal domain that binds to TBP in yeast and Drosophila.In that study, among several TBP-associated factors of this plant , none could serve as a substitute for their yeast homologs. An analysis ,136 has A. thaliana HSF1 to interact with two recombinant TBPs (TBP1 and TBP2) by affinity chromatography and an electrophoretic mobility shift assay and have uncovered the formation of complexes HSF1–TBP in vitro. In this context, TBP1 binds to HSF1 with higher affinity than TBP2 does [Drosophila heat shock gene promoter in the absence of stress [The transcriptional activation of genes encoding heat shock proteins (HSPs) is a highly conserved response to heat and other environmental stressors in all organisms ,138,139.BP2 does . By meanf stress ,141.Agrobacterium into different organisms. VirD2 also interacts with TBP in vitro and in Agrobacterium-transformed Arabidopsis cells [6-TBP.TBP not only performs an important function in the regulation of transcription but is also implicated in the control of transcription-related repair, which ensures efficient removal of DNA damage from transcribed genes . Bacteriis cells ,144; an TBP recognizes only supercoiled DNA structures similar to those seen in the TATA box when bound to TBP . SeveralSome research ,149 indiMot1 promoter [Some reports ,151 indipromoter . Mot1 acpromoter ,155. Conpromoter ,157. Motpromoter ,159. In promoter ,161. NC2promoter . NC2 repIt is known that during mitosis, when chromatin condenses, transcription is stopped and then regulated in a cell-cycle specific manner. Most transcription factors remain associated with mitotic chromosomes, but in a dynamic manner. Ref.  suggestsIt must be mentioned that, because of the high homology of a C-terminal region to that of mammalian TBP, proteins related to TBP have been identified: TBPL1 ), TLP, TBPL2, and TRF3. A good review of this topic is given in Ref. .A. thaliana participate in plant development by affecting the organization of shoot apical meristems, leaf development, flower organ and leaf formation, fertility, pollen tube growth, and the light response. Promoters of genes containing TATA boxes are occupied by the regulatory molecules that are different from those that bind to the promoters of TATA-free genes; this arrangement greatly contributes to differences in responses to internal and external signals. This means that differences in specific elements of the promoter region, in particular in the TATA box, may have dissimilar functional effects on the cell [A. thaliana genome and accurate insights into its gene expression are of great importance for both basic research and practical applications. The architecture and composition of core promoters containing the TATA box are highly dependent on the nucleotide sequence of the TATA box and on the nucleotide composition of the sequences flanking the TATA box. A large number of studies have addressed the interaction of TBP with TATA boxes and TATA-like elements in mammals and this number has begun to increase with the discovery of the influence of single-nucleotide polymorphisms in TATA sequences on phenotypic manifestations, in particular, correlations with various human diseases. Research into the interaction of TBP with TATA boxes of promoters of real plant genes, in particular A. thaliana genes, is noticeably lagging. The results of such studies will be useful for practical applications and should improve our understanding of transcription initiation in plants.Unlike most animals, plants are sedentary and cannot exit a poor-quality environment after germination. Consequently, in order to survive, they need to adapt to the specific conditions they face. In order to adapt to rapidly changing conditions, cells often regulate gene expression at the transcriptional level. At this level, elements of the TATA box affect the recruitment of cofactors and transcription factors to promoter regions of target genes. Changes in the amount of TBP in the cell . Therefo