Male infertility affects half of infertile couples and, currently, a relevant percentage of cases of male infertility is considered as idiopathic. Although the male contribution to human fertilization has traditionally been restricted to sperm DNA, current evidence suggest that a relevant number of sperm transcripts and proteins are involved in acrosome reactions, sperm‒oocyte fusion and, once released into the oocyte, embryo growth and development. The aim of this review is to provide updated and comprehensive insight into the molecular biology of spermatogenesis, including evidence on spermatogenetic failure and underlining the role of the sperm-carried molecular factors involved in oocyte fertilization and embryo growth. This represents the first step in the identification of new possible diagnostic and, possibly, therapeutic markers in the field of apparently idiopathic male infertility. Infertility is a widespread condition in industrialized countries, affecting up to 15% of couples of childbearing age . InfertiMale factor infertility accounts, cumulatively, for half of couple infertility, being solely identified in 30% of couples . NotablySpermatogenesis is a complex series of molecular events resulting in the proliferation of spermatogonial stem cells (SSCs) and in their differentiation into highly specialized, terminally differentiated spermatozoa. The development of the current high-throughput “omic” technologies has led to the recognition of the great complexity of the spermatozoon, which carries thousands of RNAs and proteins. The overall contribution of the sperm genome (including epigenetic regulation), transcriptome, and proteome to embryo formation and development needs to be investigated to identify novel molecular targets responsible for male infertility.Based on these premises, the purpose of this review is to provide updated and comprehensive insights into the molecular biology of spermatogenesis, underlining the role of the molecular factors involved in oocyte fertilization and embryo growth that are produced during spermatogenesis and carried out by the spermatozoon.Spermatogenesis, a 74-day-long process required to finally differentiate SSCs in spermatozoa, typically involves three different functional phases occurring in testicular seminiferous tubules: mitosis, meiosis, and spermiogenesis. A highly complex series of molecular events, requiring proper interactions between Sertoli cells, germ cells, epithelial tubular cells, and the integrity of the blood‒testis‒barrier (BTB), is needed for a successful spermatogenesis ,9.Sertoli cell-derived, follicle stimulating hormone (FSH)-induced growth factors ) boost Aal spermatogonia differentiation into A1 . By mitoPreleptotene spermatocytes undergo a 16-day-long meiotic division to differentiate into secondary spermatocytes. During prophase I, the first step of meiosis, double-strand breaks (DSBs) (leptotene), pairing of homologue chromosome (zygotene), and crossing over (CO) (pachytene) take place, all relying on fine molecular mechanisms. Accordingly, several proteins with enzymatic activity are expressed in this step (such as PLK-4) to ensure proper chromosome segregation, but also SPO11β, RPA1, RPA2, RPA3, MEIOB, RAD51, SPATA22, MSH4, MSH5, TEX11, TEX15, SYCE, HSF2, HEI10, RNF212, and CNTD1, required for proper CO recombination and DSBs repair .After CO has been completed, chromosome segregation (consisting of the homologues chromosome split up) occurs. It requires the presence of intact intercellular bridges, which are structures important for germ cell communication, synchronization, and chromosome dosage compensation in haploid cells. TEX14 is a structural component of intercellular bridges, highly conserved among mammals, which has been implicated in the pathogenesis of spermatogenetic failure (SPGF) .Secondary spermatocytes undergo meiosis II, through which sister chromatids divide to produce haploid round spermatids. Later on, DNA packaging with protamines, acrosome and midpiece formation, flagella organization, and cytoplasm expulsion are required for the differentiation of secondary spermatocytes into spermatozoa. This process is called spermiogenesis and requires 26 days. Finally, differentiated spermatozoa are highly specialized cells that must be capable of active motility to get into the female genital tract and penetrate the oocyte. Due to the limited sperm scavenger capacity and sperm’s well-known susceptibility to reactive oxygen species (ROS)-induced damage, all environmental factors leading to a perturbation of the pro- and anti-oxidant balance can potentially interfere with the sperm fertilization capacity ,12.The molecular factors involved in spermatogenesis whose mutations have been called into play in the pathogenesis of SPGF are shown in Fertilization is a multistep process starting once the spermatozoon has reached the oocyte. It requires the following events: capacitation, hyperactivation, acrosome reaction, binding of the spermatozoon to the zona pellucida (ZP), penetration of the ZP, fusion of the sperm with the oocyte plasma membrane, and oocyte activation .An acrosome is a vesicle under the sperm membrane and above the nucleus, in the anterior part of the sperm head, that contains pivotal enzymes involved in sperm penetration into the oocyte ,15. The ZP is a highly specialized extracellular matrix surrounding the oocyte, containing three major glycoproteins: ZP1, ZP2, and ZP3. ZP1‒3 receptors are expressed in the sperm head membrane. Their binding to ZP proteins is crucial for the sperm‒oocyte recognition phase, which follows the AR . After rAfter sperm penetration into the oocyte, male and female pronuclei lie in the same cytoplasm and a series of events happen to make them fuse together. In particular, chromatin remodeling (involving histone incorporation), genome-wide DNA demethylation and remethylation, histone modification, establishment of open chromatin patterns, and changes in chromatin conformation take place in parental nuclei . Sperm pThe fusion of male and female pronuclei forms the zygote. It will develop into a morula and then a blastocyst in about six days. The disappearance of the ZP surrounding the blastocyst follows implantation into the uterine wall, leading to the formation of ectoderm, endoderm, and mesoderm layers, needed to make a competent, functional, and independent organism .Human spermatozoa contribute to many fundamental moments for human fertility  through their genome, transcriptome, and proteome, whose products are transported inside the oocyte at the time of fertilization.A wide range of somatic gene variations has recently been called into play as possible causes of apparently unexplainable spermatogenetic failure. Mainly, these are genes involved in pivotal steps of spermatogenesis, such as germ cell proliferation, meiosis, and spermiogenesis . Their eBeyond somatic gene abnormalities, the quality of the sperm genome is of great importance for the pregnancy outcome. The sperm genome is highly packaged by protamines, which replace histones during spermatogenesis. This process, which is typical of spermatozoa, is called protamination and ensures significant DNA compaction, resulting in protection from external insults and gene silencing . AbnormaProtamination involves ~85% of sperm DNA, whereas the other ~15% remains bound to histones. This amount of histone-bound DNA is not randomly distributed, but is placed in sites playing a structural role  or in promoter regions of genes involved in embryo development . Post-trThe exposure to pro-oxidant factors can concomitantly damage both sperm DNA integrity and gene expression by inducing epigenetic modifications. Accordingly, ROS have been found capable of altering H19‒Igf2 methylation (by reducing H19 and increasing Igf2 methylation pattern) . MoreoveFinally, in addition to sperm DNA fragmentation (whose role is well recognized), sperm global methylation or methylation of imprinted genes  might alMature spermatozoa have generally been seen as a simple carrier of the paternal genome. Indeed, due to the chromatin compactness, it was believed that these cells were totally incapable of gene transcription and translation. However, some hypo-methylated DNA regions  allow access to the transcription apparatus. Furthermore, despite the majority of sperm RNA probably being transcribed prior meiosis, some evidence has shown that transcription occurs in postmeiotic spermatozoa ,37. SincThe involvement of sperm transcriptome in the pregnancy outcome has been shown in mice. Accordingly, the cleavage of sperm RNAs by treatment with a RNAse drastically decreased the rate of blastocyst formation and the live birth rate of embryos produced by intracytoplasmic sperm injection (ICSI) compared to those obtained with untreated spermatozoa . In addiSpermatozoa carry thousands of different RNAs—such as messenger RNA (mRNA), micro-RNA (miRNA), interference RNA (iRNA), antisense RNA and long non-coding RNA—and more than 4000 different mRNAs . Human sIGF2 is the first historically characterized imprinted gene whose expression depends on H19 methylation. While in the maternal allele H19 unmethylation prevents the accession to the IGF2 enhancer, H19 methylation leads to IGF2 expression in the paternal one . InteresSeveral studies have analyzed the sperm proteome; this has resulted in the characterization of 6871 proteins so far . These pn = 381), chromatin remodeling proteins acting on DNA methylation (n = 25) or histone post-translational modification (n = 118), proteins involved in non-coding RNA processing (n = 36) [GO annotation also provided evidence for the presence of 560 sperm proteins that are able to modulate gene expression. They are subdivided into transcription factors ((n = 36) . Further(n = 36) . FinallyThe metabolome includes all the metabolites, regardless of their biochemical nature, involved in biological processes. As several metabolic reactions are needed for human spermatogenesis to occur properly, the study of sperm metabolome  has captured the attention of scientists in recent years, since it may aid in the understanding of apparently idiopathic infertility.Amino acids, biogenic amines, and lipids represent the majority of the sperm metabolome, and have been found to be related to conventional sperm parameters. In more detail, the nonessential amino acid glutamine (Gln), which has been reported to be the most abundant amino acid in both spermatozoa and seminal plasma, correlates significantly with sperm concentration and motility . ArgininIn addition to amino acids, sperm lipid content has been associated with progressive sperm motility. Furthermore, the sperm concentration of total, HDL and LDL cholesterol, and triglycerides is higher in patients with oligo-astheno-teratozoospermia compared with normozoospermic men .These findings may help to address forms of apparently idiopathic male infertility and to design targeted therapies in the future. Studies investigating differences in the sperm metabolome between fertile and infertile patients are warranted.Recent advantages in the field of sperm “omics” are leading to a new understanding of the role of spermatozoa in fertility. Consequently, based on the evidence reviewed in this article, spermatozoa are not only simple transporters of their DNA within the oocyte, but also carry a wide range of important molecules  that are useful for supporting embryo mitosis.We suggest that the role of paternally-expressed imprinted genes should not be underestimated since transcription may potentially occur in the spermatozoon. Accordingly, these genes display a low-methylated pattern in their promoter region, which is consistent with transcription. This hypothesis is supported by the expression of IGF2 mRNA into human spermatozoa , and theIn addition to IGF2, several other sperm-carried factors have emerged from this “omic” analysis. In the near future, targeted study of the role of such molecules in human fertility will be needed to assess their levels in fertile and infertile patients—to be further supported by data collected in assisted reproductive techniques (ARTs) centers, prior to their becoming available (if supported by research findings) in clinical practice.In conclusion, several lines of evidence support a role for human spermatozoa in fertilization that goes beyond its DNA. A number of transcripts and proteins are transported within the oocyte and impact on embryo growth and development. Their characterization will likely bring about the recognition of novel diagnostic and, possibly, therapeutic targets in what is currently considered idiopathic male infertility.


Formation of the 3′end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave the pre-mRNA, add a poly(A) tail, and regulate transcription by protein dephosphorylation. Cleavage and polyadenylation specificity factor (CPSF) in humans, or cleavage and polyadenylation factor (CPF) in yeast, coordinates these enzymatic activities with each other, with RNA recognition, and with transcription. The site of pre-mRNA cleavage can strongly influence the translation, stability, and localization of the mRNA. Hence, cleavage site selection is highly regulated. The length of the poly(A) tail is also controlled to ensure that every transcript has a similar tail when it is exported from the nucleus. In this review, we summarize new mechanistic insights into mRNA 3′-end processing obtained through structural studies and biochemical reconstitution and outline outstanding questions in the field. In the nucleus, these include regulation of transcription as well as extensive cotranscriptional processing of precursor messenger RNAs (pre-mRNAs). Pre-mRNA processing in eukaryotes includes 5′capping with a 7-methylguanosine cap, removal of intronic sequences by the splicing machinery, and 3′-end processing . All thr3′-end processing of eukaryotic pre-mRNAs includes a cotranscriptional endonucleolytic cleavage event at a specific site in the pre-mRNA . This reMost of our understanding of pre-mRNA 3′-end processing stems from studies of human and budding yeast systems, and the general mechanisms are highly conserved across eukaryotes. Over 80 proteins have been identified as part of the eukaryotic 3′-end-processing machinery, either directly involved in cleavage and polyadenylation reactions or coordinating them with other nuclear processes .3‘-end processing in eukaryotes requires many protein factors that recognize cis-regulatory elements surrounding the cleavage site . In part‘-end-processing reaction. We next provide an overview of how pre-mRNA 3′-end processing is regulated and coordinated with other nuclear processes, particularly transcription and splicing. Finally, we highlight outstanding questions in the field of pre-mRNA 3′-end formation that remain to be addressed.In this review, we first discuss the current mechanistic understanding of pre-mRNA 3′-end processing in eukaryotes, which has been greatly elaborated on by recent studies using structural biology and biochemical reconstitution of the 3 22.1Early studies aiming to understand the mechanistic basis of cleavage and polyadenylation of eukaryotic pre-mRNAs suggested that 3‘-end processing takes place within large protein complexes specifically assembled on the pre-mRNA substrate , 14. FraThe subunits of the pre-mRNA 3′-end-processing machinery are highly similar across eukaryotes, highlighting their conserved function. Interestingly, the affinities between some components differ between yeast and humans, potentially allowing more regulation in multicellular eukaryotes . Human C2.1.1The polymerase module, known as mammalian polyadenylation specificity factor (mPSF) in humans, is a structural scaffold for CPSF/CPF , 9. It rmPSF contains four protein subunits: CPSF160, WDR33, hFip1, and CPSF30 . Human PAP is not constitutively associated with the mPSF complex, but interestingly, Pap1 is a stable subunit in yeast. Structural analyses of both the human and yeast complexes have revealed details of their highly conserved architecture. CPSF160/Cft1 contains three β-propeller domains which are arranged in a trefoil configuration, with two of these domains forming a binding cavity for an N-terminal helical domain of WDR33/Pfs2 . WDR33/PThe overall structures of mPSF and the polymerase module share a similar architecture but little sequence homology to the DNA damage–binding protein (DDB)1–DDB2 complex, which recognizes UV-damaged DNA, and the SF3b complex, which is part of the U2 small nuclear ribonucleoprotein (snRNP) involved in pre-mRNA splicing , 22. Thi2.1.2Sites for 3′-end processing are marked by the hexameric polyadenylation signal (PAS) sequence . The PASThe mechanism of PAS recognition by CPSF/CPF was first studied by UV cross-linking, which demonstrated that PAS RNA contacts the mPSF complex , 32. MorA recent cryo-EM structure of the yeast polymerase module bound to a nuclease module subunit, Mpe1, showed how the 5′part of the PAS is recognized  33). Th. Th33). 2.1.3Polyadenylation of cleaved pre-mRNAs is catalyzed by PAP/Pap1 using a polymerization mechanism dependent on two catalytic magnesium ions . On its Fip1 is an intrinsically disordered protein in isolation, but some parts become structured upon binding to Yth1 and Pap1 –26, 37. Interestingly, native mass spectrometry of endogenous CPF identified a population of the complex bound to two, instead of just one, copies of Pap1 and Fip1 . Recent 2.1.4The nuclease module, known as mammalian cleavage factor (mCF) in humans, hosts the 3′endonuclease enzyme CPSF73/Ysh1; a pseudonuclease, CPSF100/Cft2; and a third nonenzymatic subunit . In humaCPSF73/Ysh1 and CPSF100/Cft2 contain metallo-β-lactamase and β-CASP domains . The actThe trimeric complex containing CPSF73, CPSF100, and symplekin is also found within the histone pre-mRNA 3′-end-processing complex, where it is known as the histone cleavage complex (HCC) , 41. In mCF/nuclease module is connected to mPSF/polymerase module through the interaction of a highly conserved peptide of CPSF100/Cft2, called an mPSF-interacting motif (PIM), with a surface on CPSF160/Cft1 and WDR33/Pfs2 , 33. EleCPSF73/Ysh1 adopts an inactive closed conformation in most of the structures that have been determined . The 3′e2.1.51S2P3T4S5P6S7 repeats on the CTD of Pol II  and Glc7 [protein phosphatase 1 (PP1)], but these proteins are not constitutively associated with CPSF. Nevertheless, SSU72 and PP1 both copurify with an RNA-bound 3′-end processing machinery, and they perform conserved functions in coordinating transcription with 3′-end processing , 46. BotA distinct protein complex called associated with Pta1 (APT) mediates transcription termination of noncoding small nuclear (sn)RNAs and small nucleolar (sno)RNAs in yeast . APT conIn humans, 3′-end processing of snRNAs is executed by the Integrator complex, which does not share homology with APT . IntegraThe structure of the active RNA-bound Integrator complex associated with a paused Pol II elongation complex revealed that activation of the endonuclease subunit INTS11 requires the transcription elongation factor Spt5  62). In. In62). 2.2The lack of specific and efficient endonuclease activity by the CPSF/CPF complex in isolation implied that the 3‘endonuclease requires an activation step. This could prevent nonspecific cleavage of RNAs in the nucleus and enable regulation of cleavage-site selection. Many potential regulatory factors have been identified using partially purified fractions of either mammalian or yeast nuclear extract –65. Subs2.2.1CFIA is essential for the endonuclease activity of yeast CPF . It is cCStF and CF IA bind G/U-rich elements located downstream of the cleavage site. RNA binding is mediated by the RRM domains of CStF64/Rna15, which have been shown to have a preference for G/U-rich sequences in vitro . InteresCFIIm may further increase the sequence specificity of the 3′processing machinery by binding to the G-rich element found on some pre-mRNAs further downstream of the G/U-rich motif . Pcf11 pOverall, CF IA/CStF and CFIIm are required to activate the 3′-end-processing endonuclease, but the mechanism of activation remains unknown.2.2.2Yeast Mpe1 is part of the CPF nuclease module and is required for cleavage activity of the CPF complex . The humRBBP6 and Mpe1 contain a ubiquitin-like domain (UBL), a ZnF domain, and a RING finger domain. Human RBBP6 also carries a long, largely disordered C-terminal extension that is not required for activation of cleavage but may interact with transcription factors and splicing regulators –78. The A recent cryo-EM structure of the yeast polymerase module bound to Mpe1 and PAS RNA revealed that a different part of Mpe1 interacts with the Pfs2 subunit and, surprisingly, also contacts the PAS  33). Th. Th33). Mpe1/RBBP6 is likely to cooperate with the previously described cleavage factor complexes in activating the endonuclease of CPF/CPSF, but little is known about how this could be achieved. Now that the 3‘endonuclease activity has been reconstituted in vitro with both yeast and human proteins and the factors required for this reaction have been identified, the next step is to determine the mechanistic and structural details of how CPSF, RBBP6, CStF, and CFIIm carry out the endonucleolytic cleavage of pre-mRNAs. Interestingly, Mpe1 is also predicted to bind to the poly(A) polymerase Pap1, and mutations in the PSR of Mpe1 lead to hyperpolyadenylation of a cleaved pre-mRNA substrate in vitro . Given i2.2.3Additional cleavage factor proteins in both yeast and humans play regulatory roles in pre-mRNA 3‘-end processing. These factors tend to be specific to each species, suggesting that, while the basal cleavage and polyadenylation machinery is highly conserved, it may be regulated differently in yeast and humans . Yeast CIn humans, the tetrameric CFIm complex contains two copies of the RNA-binding protein CFIm25 and two copies of either CFIm68 or CFIm59 . CFIm fa2.3The 3′-end processing machinery controls polyadenylation so that all poly(A) tails are synthesized to a relatively uniform length. The median length of a poly(A) tail of a mature mRNA upon nuclear export is species specific, varying between ~60–80 adenosines in budding yeast and ~250 adenosines in humans . Hyper- Nuclear poly(A)-binding proteins, including Nab2 and PABPN1, contribute to normal control of poly(A) tail length. Once a poly(A) tail reaches ~60 adenosines in yeast and ~250 adenosines in humans, Nab2 and PABPN1, respectively, are thought to bind and inhibit processive polyadenylation , 85. In Even in the absence of poly(A)-binding proteins, polyadenylation by yeast CPF can be restricted to ~ 100–200 nucleotides in vitro and in cells . The mec2.4Not all protein-coding mRNAs in eukaryotes are polyadenylated. 3′-end processing of metazoan replication-dependent histone pre-mRNAs involves endonucleolytic cleavage downstream of a conserved stem loop structure bound by the stem loop–binding protein (SLBP). SLBP is upregulated prior to the onset of the S phase, when genes encoding replication-dependent histones are transcribed . SLBP pr3′-end processing of replication-dependent histone pre-mRNAs is executed by a specialized ribonucleoprotein machinery. The histone pre-mRNA 3′-end-processing complex contains a seven subunit Sm ring bound to U7 snRNA; subunits Lsm10, Lsm11, FLASH, and SLBP; and the HCC subcomplex that is composed of the same subunits as mCF, including endonuclease CPSF73 , 89. CStA recent cryo-EM structure of the active histone pre-mRNA 3′-end-processing machinery revealed large-scale conformational rearrangements within the HCC complex. The activation mechanism involves the opening of the CPSF73 active site due to the metallo-β-lactamase and β-CASP domains pivoting away from each other  45). Th. Th45). 3Approximately 70% of protein-coding genes in both budding yeast and metazoans produce several mRNA isoforms that differ in the sequence of their 3′ends . AlthougAPA can change the identity of the protein product if cleavage occurs before the stop codon. This can, for example, produce either a protein lacking its C-terminal regions, often affecting its function, or a truncated, nonfunctional polypeptide. In contrast, APA after the stop codon does not alter the amino acid sequence of the translated polypeptide but instead changes the length and sequence of the 3′UTR of the mRNA with potential consequences for its stability and localization. For instance, alternative mRNA isoforms with altered 3′UTR lengths often display different decay rates in the cytoplasm.Importin β1 and Bdnf) have isoforms containing longer 3′UTRs with preferential localization to neuronal projections compared to the cell body  recognized by RNA-binding proteins that target mRNAs for deadenylation and subsequent decay . Therefoell body . Recentlmembrane .APA has been covered in several excellent reviews, and we do not discuss it in detail here , 3. In bThe kinetic model would likely have a global effect on cleavage sites. For example, mRNAs in proliferating cells generally have shorter 3′UTRs, while in differentiated cells, distal PAS sequences tend to be used more often . In agre′-processing factors  control their own expression by APA, resulting in a negative autoregulatory feedback loop , have evolved mechanisms to interfere with 3′-end processing of host transcripts. Infection with influenza or HSV-1 causes transcriptome-wide inhibition of 3′-end processing leading to defects in transcription termination and pervasive read-through transcription , 118. SuBoth NS1 and ICP27 directly interact with the CPSF complex, but their inhibitory mechanisms do not seem to be conserved. NS1 binds ZnF2 and ZnF3 of CPSF30 and may compete with PAS recognition, preventing CPSF recruitment to pre-mRNAs . ICP27 h44.1′-end cleavage caused by a variety of perturbations all result in transcription read-through ..′-end clA prevailing model of transcription termination in eukaryotes posits that Pol II changes into a termination-competent complex after the PAS has been transcribed . This allosteric change likely causes Pol II to slow or pause. Subsequent cleavage of the 5′-capped pre-mRNA by CPSF/CPF and its release from the transcribing Pol II leaves an unprotected 5′phosphate on the nascent RNA, which is then targeted for degradation by a processive 5′-to-3′torpedo exonuclease, XRN2. The reduced rate of Pol II progression along the DNA template allows XRN2 to catch up with the polymerase and displace it from chromatin, terminating transcription (the torpedo model). The 3′-end-processing machinery is critical both for the deceleration of the transcribing polymerase and for providing access to XRN2 . The PNUA major outstanding question is how the CPSF/CPF phosphatase activities are triggered after transcription of the PAS. The PNUTS–PP1 complex is strongly enriched on the chromatin regions around annotated 3′cleavage sites, suggesting that specific recruitment of PNUTS–PP1 by the fully assembled 3′-end-processing complex may lead to dephosphorylation of Spt5 around the site of 3′-end cleavage . HoweverThe cleavage and polyadenylation machinery may also have roles in transcription termination beyond mediating Pol II deceleration and XRN2 accessibility . Coimmun4.2A decrease in 3′-end processing efficiency by various perturbations, such as heat and osmotic shock, as well as viral infections, leads to transcriptional read-through due to concomitant defects in transcription termination. Read-through transcription can produce a unique class of noncoding RNAs called downstream of gene (DoG) transcripts . These cIt remains unclear how general stresses such as hyperosmosis or heat shock impair or alter the function of the 3′-end-processing machinery to induce DoGs. Experimental depletion of both canonical factors and Integrator subunits has been shown to produce DoGs from proteincoding genes. Interestingly, hyperosmosis reduces the association between transcribing Pol II and the Integrator complex, while its interactions with canonical 3′-end-processing factors remain unaffected . Since tThe functional consequence of DoG transcription also remains unclear. Since DoGs tend to remain associated with chromatin, it has been hypothesized that DoG production may affect the three-dimensional organization of chromatin, and DoGs themselves may help reinforce the structure of the nucleus by interacting with nuclear scaffold proteins . Future 4.3If read-through transcription extends into a neighboring gene, the rate of transcription at this next gene might be reduced due to transcriptional interference , 131. MeGenes are more closely spaced in the yeast genome than in the human genome , where t4.4Recent advances in nascent RNA sequencing methods have revealed unexpected coordination between cotranscriptional splicing and 3′-end processing of eukaryotic pre-mRNAs. Long-read sequencing of chromatin-associated RNA has enabled the elucidation of full-length pre-mRNA processing intermediates, allowing unambiguous determination of their splicing status and the position of Pol II along the gene at the time of sample preparation –136. TheHow splicing stimulates 3′-end processing remains unknown. It has been suggested that splicing alleviates constitutive inhibition of 3′-end cleavage. A candidate inhibitor of 3′-end cleavage is the U1 snRNP that is bound to the 5′splice site of introns. U1 snRNP interacts with transcribing Pol II and also prevents intronic polyadenylation at cryptic PAS sites by inhibiting the assembly of an active 3′-end-processing complex. The removal of U1 snRNP after successful splicing of the 3′-terminal intron may then allow efficient 3′-end cleavage , 137. OtRead-through transcripts that remain unspliced are generated at low levels even in unperturbed cells in the absence of external stress . Such un53‘-end processing of pre-mRNAs plays a critical role in gene expression in eukaryotes. The relatively simple cleavage and polyadenylation reactions require at least a dozen polypeptides. This rather elaborate machinery ensures the fidelity and specificity of 3‘-end processing. In vitro reconstitution and structural studies using a variety of techniques have provided a glimpse into the architecture and mechanism of the 3′-end-processing machinery in both yeast and humans . However


How a single genome can give rise to distinct cell types remains a fundamental question in biology. Mammals are able to specify and maintain hundreds of cell fates by selectively activating unique subsets of their genome. This is achieved, in part, by enhancers—genetic elements that can increase transcription of both nearby and distal genes. Enhancers can be identified by their unique chromatin signature, including transcription factor binding and the enrichment of specific histone post-translational modifications, histone variants, and chromatin-associated cofactors. How each of these chromatin features contributes to enhancer function remains an area of intense study. In this review, we provide an overview of enhancer-associated chromatin states, and the proteins and enzymes involved in their establishment. We discuss recent insights into the effects of the enhancer chromatin state on ongoing transcription versus their role in the establishment of new transcription programmes, such as those that occur developmentally. Finally, we highlight the role of enhancer chromatin in new conceptual advances in gene regulation such as condensate formation. Enhancers were initially defined not by their genomic features, but by their ability to enhance transcription irrespective of distance, position and orientation relative to a target gene . Enhancein vivo and may have additive effects on their target genes ), are generally considered to facilitate H3K4me3 at promoters under various contexts [Drosophila Trithorax-related [Trr]) are responsible for the majority of H3K4me1 at enhancers ,70b) . . trans . I. Icis inESCs (b) ); or (iiESCs (b)  and anotESCs (b) . Taken t6.a). Bromodomain-containing proteins are anchored to chromatin through their association with acetylated lysine, and then in turn act as scaffolding proteins to recruit additional transcriptional machinery. For example, BET proteins through their extra terminal (ET) domain recruit the positive elongation factor b (P-TEFb) to promoters, resulting in cyclin-dependent kinase 9 (CDK9) phosphorylation of RNAPII and pause release [As mentioned above, one likely function of enhancer acetylation, both on histones and transcription factors, is to serve as a scaffold for recruitment of transcription machinery to chromatin. Acetylated lysines mainly on histone, but also non-histone proteins, are recognized by bromodomains, which are present in a diverse array of transcriptional coactivators . These ifigure 1 release –173. The release –176 resu release ,177.In addition to bromodomains, both tandem plant homeodomain (PHD) zinc fingers and Yaf9, ENL, AF9, Taf14, Sas5 (YEATS) domains have recently emerged as additional classes of lysine acetylation readers –180. Whi7.a). Such structures are hypothesized to form hubs where multiple enhancers and promoters converge resulting in a high frequency of transcription with low variation [Although there are thousands of enhancers in any given cell type with signatures of active chromatin, a subset of enhancers display particularly high density of transcription factor binding, are highly enriched with cofactors such as CBP/p300, resulting in high levels of H3K27ac, and are bound by high levels of chromatin regulators such as MED1 ,188. Thefigure 3ariation .Figure in vitro and condensate formation in cells [a).Biomolecular condensates are thought to form through weak, multivalent, and dynamic interactions that can be facilitated by intrinsically disordered regions (IDRs) in proteins. Such IDRs are common in the activation domains of transcription factors as well as coactivators such as MED1 and BRD4, and these regions have been shown to promote LLPS in cells –198. In in cells  .In addition to IDR-based non-traditional protein–protein interactions, structurally based weak multivalent interactions may contribute to the formation of nuclear condensates. Interestingly, linked domains resulting in multivalent engagement are a common feature of chromatin-associated proteins and complexes . For exaome core . StrikinUltimately, what is the purpose of condensate formation at cell-type-specific enhancers? A number of studies demonstrate that RNAPII assembles at condensates in cells –203, and8.In the past decade, major advances in DNA sequencing technology and computational tools have made it possible to identify a wide variety of epigenomic features genome-wide. However, identifying the function of each feature remains a challenge due to widespread cross-talk between histone variants, modifications and nucleosome depletion. For example, loss of either MLL3/4 activity or H3.3 deposition reduces H3K27ac levels without any clear effect of H3.3 on MLL3/4 activity. Together with the observation that targeted recruitment of dCas9-p300 to silent enhancers and promoters is sufficient for gene activation , these dEmerging technologies will no doubt remain key to understanding enhancer biology. Novel imaging techniques will reveal the spatio-temporal dynamics of enhancer–promoter interactions in living cells, allowing greater understanding of how and when proximity is required developmentally ,212. Imp


In embryos treated with EGCG, both gene expression and histone acetylation  returned to basal levels, and the phenotype was recovered. As shown by the results, the histone hyperacetylated landscape promoted by BPA in the sperm alters the chromatin structure of the progeny, leading to the overexpression of the histone acetyltransferase and genes involved in cardiogenesis.Parental exposure to bisphenol A (BPA) has been linked to a greater incidence of congenital diseases. We have demonstrated that BPA induces in zebrafish males an increase in the acetylation of sperm histones that is transmitted to the blastomeres of the unexposed progeny. This work is aimed to determine whether histone hyperacetylation promoted by paternal exposure to BPA is the molecular mechanism underlying the cardiogenesis impairment in the descendants. Zebrafish males were exposed to 100 and 2000 µg/L BPA during early spermatogenesis and mated with non-exposed females. We analyzed in the progeny the expression of genes involved in cardiogenesis and the epigenetic profile. Once the histone hyperacetylation was confirmed, treatment with epigallocatechin gallate (EGCG), an inhibitor of histone acetyltransferases, was assayed on F1 embryos. Embryos from males exposed to 2000 µg/L BPA overexpressed the transcription factor  The worrmations .Regarding the transmission of the effects to the next generation, it has been noticed that both maternal  and pateMale germ cells undergo a unique epigenetic remodeling throughout the spermatogenesis, which consists on the acquisition of different covalent modifications in DNA or DNA-associated proteins that regulate gene expression without changing the genetic code ,25. ShouEpigallocatechin gallate (EGCG), the major polyphenolic compound of green tea (7.1 g EGCG per 100 g green tea leaves), has been found to exhibit a large number of therapeutic properties for heart health: antioxidant, anti-inflammatory, anti-atherosclerosis, and anti-cardiac hypertrophy ,37. SincDanio rerio) males were exposed to BPA during early spermatogenesis and F1 embryos were analyzed to evaluate the epigenetic profile and the expression of candidate genes related to cardiogenesis. Once the epigenetic changes were established, a rescue treatment with EGCG was assayed on the embryos.In this scenario, our hypothesis is that the cardiotoxic effects promoted by paternal BPA exposure are triggered by changes in the histone acetylation pattern. To confirm this hypothesis, zebrafish (esr2a (estrogen receptor beta), gper1 (G protein-coupled estrogen receptor), and esrrga (estrogen-related receptor gamma a) was not altered in F1 embryos at 3.3 hpf after paternal BPA exposure (esr2b (estrogen receptor beta) was overexpressed in 24 hpf embryos obtained from males treated with both doses of BPA  transcription factor, ed males C.esr2b and hand2, as well as in that of kat6a , we anae of BPA D. hand2 and esr2b, the DNA methylation of a CpG island in the exon 1 of esr2b and in the promoter of hand2—were CpG islands are located (UCSC Genome Browser)—was analyzed in 24-hpf-embryo obtained from control and BPA-exposed males and also when EGCG treatment was applied to F1 embryos. The results showed that the DNA methylation of these two regions did not change either in F1 embryos obtained from males exposed to BPA or in F1 embryos treated with EGCG  specifically approved by the University of León Bioethical Committee as well as by the competent body of Junta de Castilla y León (project number: ULE009-2016). All the animals were manipulated in accordance with the Guidelines of the European Union Council , following Spanish regulations  for the use of laboratory animals.®).Eight-month-old zebrafish, AB strain (wildtype), were maintained in 1.5 L aquaria  with a recirculating water system . Animals were fed twice a day with dry food ) or 100 and 2000 μg/L BPA  for two weeks, and the last week they were maintained in 1.5 L water. This schedule allows that, by the time of mating, mature spermatozoa have been exposed to BPA only during early spermatogenesis but it has not affected the cells during meiosis or spermiogenesis, which together last approximately one week in zebrafish [Adult males were exposed to ethanol and BPA in groups of four in glass tanks containing 1.5 L water. To carry out the experiments, males were maintained in water with the vehicle  methylene blue at 28 °C in darkness until different probes were done. Embryo mortality was evaluated from 3.3 hpf each day until they reached 120 hpf. At 7 seven days post fertilization, phenotype of control and exposed larva were assessed under a stereomicroscope (Leica MZ16F). Transcriptomic and epigenetic analysis were performed at 3.3 and 24 hpf.After these 3 weeks, each of the four males per tank were mated with non-exposed females , giving a total of 32 mating groups per treatment, which allow obtaining the large number of embryos required to perform all the analytical techniques (three replicates per technique). Gene expression, histone acetylation marks, as well as acetylation and methylation of promoters were analyzed. In order to explore the potential reversion of paternal BPA effects using an inhibitor of histone acetylation, F1 embryos from control males and males exposed to 2000 μg/L BPA, were split up in two samples, being one of them treated with 50 µM EGCG from fertilization to blastula stage  . This doExpression of candidate genes, related to estrogen signaling and to cardiogenesis, was analyzed in F1 embryos from control and BPA-exposed males, treated and non-treated with EGCG. RNA was extracted from a pool of 30 F1 embryos at 3.3 and 24 hpf, using Trizol reagent according to manufacturer’s protocol. The concentration of RNA samples was measured using the NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Scientific), whereas the integrity was assessed by electrophoresis on agarose gel (data not shown).esr2a, esr2b, esrrga, gper1), the histone acetyltransferase (kat6a), the heart transcription factors (hand2 and gata5) and the housekeeping genes (18S rRNA for 3.3 hpf-embryo and actb2 for 24 hpf-embryo) were the same as those detailed by our group [gata4 and bmp4 are described in esr2a, esr2b, esrrga, gper1 [kat6a was evaluated since our previous results showed that paternal exposure to 2000 μg/L BPA led to an overexpression of this gene in F1 embryos at 24 hpf [The cDNA syntheses and RT-qPCR conditions as well as zebrafish primer sequences for the estrogen receptors  and of t 24 hpf . w/v) PFA overnight at 4 °C. From this moment onwards, the protocol described by our group for the detection of H3K9ac  and H3K27ac  was followed [®488 relative to the nuclear area stained with DAPI by using Image J software of, at least, 200 cells per replicate.For the whole mount immunostaining, 5 F1 control embryos and embryos treated with EGCG were collected at 3.3 hpf for each replicate, when changes in histone acetylation triggered by BPA where noticed , and fixfollowed . The potfollowed . Negativkat6a, esr2b, and hand2 at 24 hpf, when the expression of these genes was evaluated. An intergenic region of the genome lacking acetylation has been used to prove the absence of non-specific bindings. The same qPCR conditions and primer sequences as those described in our previous work [Chromatin immunoprecipitation (ChIP) was performed in pools of 150 embryos at 24 hpf for each replicate and treatment following the protocol of Santos-Pereira and colleagues  to assesous work  were useAfter DNA isolation from pools of 30 embryos at 24 hpf for each replicate and treatment, it was quantified using the NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Scientific) and 600 ng of genomic DNA were treated with sodium bisulfite using EZ DNA Methylation™ Gold kit (Zymo Research), to selectively turn non-methylated cytosines into uracils. Then, total PCR was performed from 1 µL bisulfite-converted DNA and products were diluted 1:20 for the nested PCR. Programs for both total and nested PCR were: phase of 2 min at 94 °C followed by 40 cycles of 94 °C denaturation for 25 s, 58 °C annealing for 1 min and 72 °C extension for 2 min, and an extra step at 70 °C for 7 min. Then, 20 µL of products from nested PCR were mixed with annealing buffer and streptavidin beads. Once products were denaturated and annealing of pyrosequencing primers was achieved, final reactions were run with the PyroMark Q96 ID (Qiagen), which was also used for the analysis of CpG methylation that was calculated as the ratio of peak heights in the pyrogram. https://genome.ucsc.edu/, accessed on 9 January 2021). The primers for total PCR, nested PCR, and pyrosequencing were designed using MethPrimer and they are listed in The sequence of the CpG islands near the transcription start site (TSS) were obtained from UCSC Genome Browser . For parametric data one-way ANOVA followed by DMS post hoc test was used, whereas for non-parametric data a Kruskal–Wallis test was applied following pairwise comparison and significance adjusted with Bonferroni correction. The type of test performed for each analysis as well as In vivo zebrafish male exposure allowed us to find out the molecular mechanisms by which paternal exposure to BPA exerts long-term effects on F1 cardiogenesis: male exposure induces an increase in sperm histone acetylation that is inherited by the F1, altering the chromatin structure of crucial genes for heart development and that of the HAT in charge of maintaining the profile. Moreover, when F1 embryos obtained from BPA-exposed males were treated with EGCG, histone acetylation levels were restored, avoiding the overexpression of estrogen receptors and transcription factors and, therefore, reducing the probability of cardiac disease.


Drosophila development by controlling spatiotemporal properties of their activation and repression. But the exact proteins that cooperate to stall Pol II in promoter-proximal regions of developmental genes are still largely unknown. The current work describes the molecular mechanism employed by the Negative ELongation Factor (NELF) to control the Pol II pause at genes whose transcription is induced by 20-hydroxyecdysone (20E). According to our data, the NELF complex is recruited to the promoters and enhancers of 20E-dependent genes. Its presence at the regulatory sites of 20E-dependent genes correlates with observed interaction between the NELF-A subunit and the ecdysone receptor (EcR). The complete NELF complex is formed at the 20E-dependent promoters and participates in both their induced transcriptional response and maintenance of the uninduced state to keep them ready for the forthcoming transcription. NELF depletion causes a significant decrease in transcription induced by 20E, which is associated with the disruption of Pol II elongation complexes. A considerable reduction in the promoter-bound level of the Spt5 subunit of transcription elongation factor DSIF was observed at the 20E-dependent genes upon NELF depletion. We presume that an important function of NELF is to participate in stabilizing the Pol II-DSIF complex, resulting in a significant impact on transcription of its target genes. In order to directly link NELF to regulation of 20E-dependent genes in development, we show the presence of NELF at the promoters of 20E-dependent genes during their active transcription in both embryogenesis and metamorphosis. We also demonstrate that 20E-dependent promoters, while temporarily inactive at the larval stage, preserve a Pol II paused state and bind NELF complex.For many years it was believed that promoter-proximal RNA-polymerase II (Pol II) pausing manages the transcription of genes in  Drosophila cell culture1. In our previous works we have described in detail the primary stages of 20E-dependent induction and found new molecular partners of the EcR/Usp ecdysone sensor3. The present study aims to characterize the molecular complex responsible for the organization of the Pol II pause at 20E-dependent genes.Over the years, researchers have aimed to discover and characterize proteins and protein complexes that regulate the transcription of genes. To date, multiple complexes have been described, and we are now entering a breathtaking new phase of research that allows us to find a suitable place for each regulator in the complex multi-step process of eukaryotic transcription. A detailed system of model genes is required for the experimental modeling of the transcriptional process. For this purpose, we chose a set of developmental genes that can be rapidly induced by the addition of 20-hydroxyecdysone (20E) hormone in Drosophila heat-shock protein genes4. RNA polymerase II was shown to be recruited at the promoters of these genes in their inactive state, and stress-induced activation of the heat shock genes resulted in Pol II release from their promoters into the gene bodies. Subsequent research has proven that the Pol II pause is an important regulatory phenomenon for many genes in various organisms7.The phenomenon of promoter-proximal Pol II pausing (Pol II pausing) was first described in a study of 9. However, the mechanism guiding Pol II pausing at 20E-dependent promoters was so far not described. Previously, we observed the presence of Pol II on many 20E-dependent promoters in their inactive state, and showed that the induction of 20E-dependent transcription leads to a change in the degree of Pol II phosphorylation rather than an increase in the amount of Pol II associated with the promoter2. In other words, we showed that 20E-dependent genes are regulated via Pol II pausing. In a recent work we revealed an interaction between the EcR/Usp sensor and the NELF-A subunit of the Negative elongation factor (NELF) and hypothesized that the latter may be responsible for Pol II pausing at 20E-dependent promoters3.Early research indicated that some genes regulated by 20E use Pol II pausing as a mechanism for controlling their transcription10. The NELF- and DSIF-mediated transcription block is usually overcome by p-TEF-dependent phosphorylation, which is stimulated by transcriptional activators11. The NELF complex consists of four protein subunits that are evolutionarily conserved and are inherent in organisms that regulate transcription by Pol II pausing12. Currently, the described molecular functions of NELF are not limited to establishing a Pol II paused state13, and NELF was even found to be recruited to the promoters of actively transcribed genes14. The exact functions of NELF at the promoters of active genes are currently only poorly understood, but there is evidence for its role in mRNA processing and loading of the Cap-associated complex16 as demonstrated by the interaction between the NELF-E subunit and RNA in the Pol II paused complex17.NELF is the most well-known factor involved in Pol II pausing at eukaryotic genes, where it cooperates with the DSIF complex to halt Pol II elongation18. Namely, Pol II paused promoters participate in organization of 3D chromatin topology in embryogenesis by anchoring contacts coming from developmental enhancers19. It is believed that the main role of the Pol II pause in development is to synchronize transcriptional activation in individual cells of the embryo21. It was shown that the Pol II pause-derived temporal synchronization of gene activation is crucial for the coordination of cellular behavior at the most important stages of morphogenesis (e.g. during gastrulation)20. However, there is little information regarding the factors responsible for maintenance of the Pol II pause during the early hours of development. Our present study brings forth new insights by describing the role of NELF in transcriptional regulation of one of the central groups of genes in Drosophila development whose transcription is induced by 20E hormone.Pol II pausing is involved in the dynamic transcriptional regulation of developmental genes2, including the NELF-A subunit. We validated this interaction in a previous report by co-immunoprecipitation of these proteins from the prepupal nuclear extract . We also observed a decrease in the 20E-dependent transcriptional induction of some genes upon NELF-A knockdown. Here, we describe the relationships between EcR and the NELF complex in detail and propose a mechanism for NELF function at 20E-dependent genes.Recently, we identified novel protein interactors of the EcR/Usp heterodimer using in vivo proximity-dependent biotin ligationDrosophila Schneider cell line 2 (S2) cells were maintained at 25 °C in ecdysone-free Schneider’s insect medium (Sigma) containing 10% FBS (HyClone). The treatment of S2 cells with 20-hydroxyecdysone (20E)  was performed at a final concentration of 0.3 μM, as was described previously2. As different studies have employed various duration of 20E treatment to induce transcription, we limited our approach to only identify direct targets of 20E by limiting the time of 20E treatment to 1 h. This prevents primary-induced transcripts (which are in turn transcription factors) to be translated and shuttled back to the nucleus to affect transcription of their targets.22 and RNA interference-mediated knockdowns of GFP, NELF-A, B, D and E subunits of NELF complex, EcR and Spt5 in Drosophila S2 cells2 were performed as previously described. Efficiency of RNA knockdown was evaluated by qPCR and Western-blot and provided at Fig. Immunoprecipitation23. ChIP-Seq libraries were obtained using the NEBNext DNA library preparation kit (New England Biolabs). Only the library fragments of 250–500 bp were subjected to NGS sequencing. New generation sequencing was performed by Evrogen (evrogen.ru) with the Illumina NovaSeq6000 sequencer. For each of the ChIP‐Seq libraries, approximately 5–14 millions of unique paired mappable reads were obtained. The paired‐end reads in FastQ format were mapped to the Drosophila genome assembly dm6 using Bowtie224 and filtered .The chromatin immunoprecipitation (ChIP) was performed exactly as previously described25. The final bigwig files (representing the protein binding profiles) were obtained using bigwigcompare tool as ratio of ChIP signal to Input . Pile-up profiles were calculated as a median level of protein binding for the distributions of Rpb3, NELF-A, NELF-B and NELF-E near TSSs and Rpb3, NELF-A, NELF-B, NELF-E and CBP/Neijre near enhancers of 20E-dependent genes in Drosophila S2 cells. Pile-up profiles for the distributions of Rpb3, NELF-A and Pol II S2P in different stages of Drosophila development were also calculated as a median level. The CBP/Neijre binding sites in Drosophila S2 cells were defined by MACS2 with the following parameters: -gsize ‘120,000,000’ -keep-dup ‘1’-qvalue ‘0.01’ -mfold ‘5’ ‘30’ –bw ‘375’ 2 >  and 1 > macs2_stderr26. Corresponding input DNA was used as a control for peak calling.BigWig files were generated using bamCoverage 3.0.2 with scores representing number of reads normalized by the size of the library 1. In the present study , expressed both during embryogenesis and pupariation, is provided in Table A list of 20E-dependent genes (induced in S2 Schneider cells upon 1 h 20E treatment) was obtained previouslyA list of enhancers of rapidly induced 20E-dependent genes was generated based on the following criteria: presence of a CBP/Neijre peak, absence of a TSS, and location within a 20E-induced gene . We did not use any figures or text from the previously published manuscripts—only data deposited in free access databases.GRO-Seq data were described previously and deposited as GSM3274627 and GSM3274628Drosophila genome)25. The standard error was calculated and displayed on the graphs as lighter areas around the main line of the profile.Pile-up profiles for Fig. p-value < 0.01, *—p-value < 0.05. Statistical analysis was performed relative to the transcriptional induction levels of the genes in GFP dsRNA treated S2 cells.Transcriptional induction level in Fig. The correlation for the impact of NELF-A, NELF-B, NELF-D and NELF-E subunits knockdowns on transcriptional induction levels of 20E-dependent genes provided in Fig. 25. NELF-A, NELF-B, NELF-E, Rpb3, Pol II Ser2P and Pol II Ser5P binding levels were estimated as an enrichment . CBP/Neijre and FLAG-EcR binding levels were estimated as difference between ChIP-Seq signal and Input. RNA-Seq signal represent a number of reads at the corresponding position. The standard error is displayed on the graphs as lighter area around the main line of the profiles.Pile-up profiles of ChIP-Seq and RNA-Seq signals in Figs. NELF-A, NELF-B and NELF-E binding levels at the Fig. Transcriptional levels in Fig. The flies of Oregon-R-modENCODE stock were used . Embryos were collected in the fly cages for 2 h using apple juice agar plates. Then plates were incubated at the 25 °C for the required period of time. The time after egg laying (AEL) was calculated starting from the moment the agar plates were placed in the fly cages.L3 larvae, corresponding to the puff stage 1–3, were collected by culturing larvae in the fly media supplemented with 0.05% bromophenol blue to mark the guts of feeding animals (L3 larvae had dark blue gut). Prepupae corresponding to 0–1 h after puparium formation (APF) were collected according to description of this developmental stage: white motionless prepupae starting to evert their anterior spiracles.2, 15 mM HEPES pH7.6, 0,5% Triton X-100) containing 0.7% of formaldehyde for 10 min and incubated for 5 min with 125 mM Glycine. Then cells were washed for three times with homogenization buffer. The remaining ChIP protocol was performed as described previously23.For the ChIP-Seq experiments embryos were dechorionized, washed and homogenized in a buffer  were generated in this study. The epitopes for antibody production were expressed as 6 × His-tagged fusion proteins in Escherichia coli, affinity-purified on Ni Sepharose 6 Fast Flow , according to the manufacturer’s protocol, and injected into rabbits, following the standard immunization procedure. Antibodies were affinity-purified using the same epitopes as were used for immunization. The specificity of antibodies against EcR, Spt5, NELF A and NELF B was characterized in the RNA interference experiments by the specific depletion of corresponding proteins . For the ChIP-Seq analysis of CBP/Neijre, Rpb3, Spt5, NELF-A and NELF-E we used previously described antibodiesns Figs. –S4. AntiDrosophila S2 cells were shown to be suitable for the investigation of the ecdysone response as most ecdysone cascade genes are expressed upon 20E treatment1. We used this system to evaluate the influence of the 20E hormone on the interaction between EcR and NELF. We first investigated interactions between NELF subunits and EcR in Drosophila S2 nuclear extracts using specific antibodies  of 20E-dependent genes both in mock and NELF-B/NELF-E dsRNA-treated cells . We estimated the values of Pearson’s correlation for each pair of NELF subunits knockdowns and found them to be strongly positive . These promoters represent an illustrative example of a strong correlation between chromatin-bound level of NELF factor and Pol II pause (a promoter-bound level of Pol II). The hr4 promoter is in a less paused state and characterized with fewer amount of NELF and Pol II than dhr3. This may be the reason why we did not observe a change in an induced transcriptional response of the hr4 gene upon the knockdown of NELF subunits  not only at promoters but also within gene bodies. This fact did not appear during investigation of averaged NELF profiles because they were calculated using coding regions of genes while some NELF subunits were found present at the sites located in the introns. To reveal whether these sites are intronic enhancers, we compared NELF subunits profiles with ChIP-Seqs of FLAG-EcR and CBP/Neijre, the ecdysone receptor and the major histone acetyltransferase marking enhancers, respectively. We indeed observed the presence of EcR and CBP/Neijre at NELF-bound sites at the bodies of the To verify the specificity of NELF enrichment at 20E-dependent promoters and enhancers, we plotted an averaged profile of NELF binding for the transcriptional end sites (TESs) of 20E-dependent genes Fig. . We deteUpon close examination, we observed that at individual 20E-dependent promoters NELF-A binding does not exactly match binding of the NELF-B and NELF-E subunits  demonstrated both the high level of NELF binding and the rate of its decrease upon NELF subunits knockdown  as a set of control genes that have alternative developmental transcriptional profiles, as their transcription is induced during the first hours of embryogenesis and responsible for embryo segmentation  as well as represses an androgen signal‐transduction cascade by decreasing the stability of AR39.Interestingly, the mechanism of NELF functioning has been the subject of many detailed studies, while the process of its recruitment to a regulated gene is not well understood. We searched the literature to understand whether the recruitment of the NELF complex to its targets through interaction with nuclear receptors is widespread or not. It turned out that the interaction of EcR and NELF is not the only example. It was previously demonstrated that the NELF-B subunit of the human NELF complex interacts with the estrogen receptor (ER) and represses ERα-mediated transcriptionhsp70 gene. This classical Drosophila Pol II pausing model gene demonstrates three times increase in its productive transcription elongation upon NELF knockdown, which is assumed to be an evidence of NELF role in restraining of the Pol II elongation process40. However, NELF knockdown doesn’t result in the activation of the 20E genes to their fully induced transcriptional levels. NELF does not function as an active repressor of 20E-dependent genes. We suggest, that its presence at these genes in their uninduced state prepares them for the forthcoming transcription via Pol II pausing, which coordinates their response to 20E.NELF plays a dual role in the transcriptional regulation of 20E-dependent genes described in the present study. On one hand NELF participates in Pol II pausing at uninduced state of these genes, and on the other hand it is involved in their induced transcriptional response. We have demonstrated that NELF depletion causes the disruption of Pol II pause during the uninduced state of 20E genes, which is confirmed by a decrease in Pol II binding to the promoters and in the level of abortive promoters-proximal transcription. Some of 20E-inducible genes show a significant increase in their productive transcription in the uninduced state upon NELF subunits knockdown. This effect very well corresponds to the behavior of 17. NELF binds the polymerase funnel, bridges two mobile polymerase modules, and contacts the trigger loop, thereby restraining Pol II mobility that is required for pause release. All of these NELF properties are in line with its role as a pausing factor, which prepares genes for the forthcoming transcription. Much less clear is how NELF restraining of Pol II is integrated in induced transcription of the genes41.The recently described structure of the Pol II-DSIF-NELF complex has shed light on the molecular function of NELF in Pol II pausing33. Another study demonstrated that DSIF is globally required for Pol II accumulation at the promoters and for the majority of productive mRNA synthesis42. Thus, we think that the presence of NELF on the promoters of actively transcribed genes allows it to indirectly influence the elongation of Pol II, affecting the stability of the complex between DSIF and Pol II inside temporarily paused Pol II complex. Although previously observed, the effect of NELF on the stabilization of the complex between Pol II and DSIF is not well-described. Early in vitro assays investigating DSIF and NELF loading revealed that the presence of DSIF in the Pol II complex is essential for NELF binding34. But the reverse impact was also observed: in low concentrations DSIF was unable to bind Pol II in the absence of NELF. In a more recent structural study, it was shown that a NELF-A subunit ‘tentacle’ contacts the DSIF factor within the Pol II-DSIF-NELF complex17. This direct interaction actually may stabilize the complex between DSIF and Pol II.Our present work demonstrates the inability of 20E-dependent genes to reach their full inducible level upon NELF depletion. We link the given function of NELF with its role in stabilization of a complex between DSIF and Pol II in the early moments of elongation of 20E-dependent genes. It is known that in DSIF-depleted cells, a fraction of Pol II molecules is dislodged during elongation, thus decreasing the number of Pol II complexes that complete the transcription cycleAt the same time, the leading effect of NELF on the induced transcriptional response may be the stabilization of the temporary Pol II pause, which provides the necessary time for the formation of a productive elongation complex.Drosophila genome during the larva-to-pupa transition in the wing imaginal discs43. They revealed a highly dynamic EcR binding to its target sites during development. However, comparing EcR binding in wing tissues and S2 cells of embryonic origin, they observed some common EcR binding sites. The presence of a constant set of genes that are induced by 20E in various tissues types was also reported by Shlyueva and colleagues44 in a study on ecdysone-dependent enhancers. But still, the processes that occur on promoters of these constant 20E targets at different stages of development were not characterized until now.20E-dependent genes are rather unusual in that they are activated repeatedly in development. A recent study by Uyehara and McKay compared the changes in EcR binding across the 45. ChIP-Seqs of Pol II total, Pol II Ser2P and NELF during development allowed us to hypothesize a molecular mechanism driving transcription of 20E-dependent genes. According to our data, these genes are activated in embryogenesis by recruiting Pol II, retain bound Pol II on their promoters during larval stages, and are again activated in pupariation by stimulating release of the Pol II pause. We assume that NELF helps to maintain a Pol II pause at the promoters of 20E-dependent genes during Drosophila development, since NELF is responsible for the Pol II pause at 20E-dependent promoters in the cell culture. The presence of three out of four NELF subunits on 20E-dependent promoters at the larval stage, when these genes are inactive, but are preparing for the forthcoming induction, further confirms the involvement of NELF in the process of Pol II pausing.The present study deals specifically with a set of 20E constant targets, most of which are induced by 20E hormone in many cell types46. It was shown, that in Drosophila embryo development the transcriptional memory plays an important role: transcription of gene in a daughter cell is activated faster and stronger if this gene was active in a mother nucleus46. The Pol II pausing was found to be a part of a mechanism implementing transcriptional memory—the transition of genes in the Pol II paused state during embryo development increases the likelihood of their activation47. Why a cell chooses transcriptional pausing for temporal inactivation of 20E-dependent genes remains to be seen—perhaps it preserves the competence of the tissue in which the ecdysone cascade has already been activated to repeat the activation in the course of metamorphosis.The transient transcriptional pausing of 20E-dependent genes during larval development can be viewed as a type of transcriptional memory that allows genes to be easily reactivatedDrosophila development, the role of the NELF complex in these processes has mostly been overlooked48. Currently it seems that the role of this factor may go beyond spatiotemporal control of transcription activation—likely, NELF also impacts the quality of the produced transcripts, as well as contributes to transcription repression. We hope that the power of the available experimental techniques will make it possible to further dissect the role of NELF in these processes taking place at Drosophila developmental genes.Despite the multitude of previous studies on Pol II pausing mechanisms in early Supplementary Figure.