Promoters are DNA sequences where the process of transcription starts. They can work constitutively or be controlled by environmental signals of different types. The quantity of proteins and RNA present in yeast genetic circuits highly depends on promoter strength. Hence, they have been deeply studied and modified over, at least, the last forty years, especially since the year 2000 when Synthetic Biology was born. Here, we present how promoter engineering changed over these four decades and discuss its possible future directions due to novel computational methods and technology.S. cerevisiae, a popular chassis for gene circuits. We describe the initial techniques and achievements in promoter engineering that predated the start of the Synthetic Biology epoch of about 20 years. We present the main applications of synthetic promoters built via different methods and discuss the latest innovations in the wet-lab engineering of novel promoter sequences.Synthetic gene circuits are made of DNA sequences, referred to as transcription units, that communicate by exchanging proteins or RNA molecules. Proteins are, mostly, transcription factors that bind promoter sequences to modulate the expression of other molecules. Promoters are, therefore, key components in genetic circuits. In this review, we focus our attention on the construction of artificial promoters for the yeast  To th chassis . The bakhemicals ,8,9.Synthetic gene circuits are made of transcription units (TUs) that, in eukaryotes, are composed of three different standard biological parts: promoters, coding sequences (CDSs), and terminators. Inside a circuit, TUs interact via the exchange of proteins or RNA molecules encoded into their CDSs. Initiation and control of transcription occur at the promoter level. Therefore, within a genetic circuit, promoters shall be chosen and engineered carefully to obtain the necessary amount of proteins (and RNAs) that make the circuit work as desired.constitutive and inducible. Constitutive promoters provide relatively stable protein/RNA expression levels under different cell-culture conditions [Endogenous promoters are characterized by different strengths  and are divided into two classes: nditions . Inducibnditions ,12,13.S. cerevisiae over the last forty years. First, we introduce the general structure and working of promoters in budding yeasts. Then, we discuss the engineering of hybrid constitutive promoters. Finally, we illustrate the implementation of regulated promoters and the interactions with their corresponding  transcription factors (TFs). Applications of the diverse synthetic promoters are presented as well.In this review, we describe the different ways synthetic promoters have been built and used in the yeast A promoter is a DNA sequence that enables transcription initiation. The structure of a promoter is specified by the transcription start site (TSS) and the position, relative to the TSS, of various protein-binding sequences. To design promoters for synthetic biology, it is essential to understand how the promoter structure influences promoter strength and performance .S. cerevisiae RNA polymerase II-dependent promoters are characterized by three main elements: the upstream activating sequence (UAS), the TATA box, and the transcription start site [S. cerevisiae do not have a TATA box and are recognized and bound by RNA polymerase II via other structural features (such as a DNA bend) [TATA-less promoters are out of the scope of this review since they have been used seldom in synthetic gene circuits.art site . Each ofNA bend) . HoweverS. cerevisiae, like the enhancers in higher eukaryotes, are bound by activator proteins that recruit to the promoter RNA polymerase II together with the general transcription factor [core promoter, which starts with the first TATA box and finishes at the end of the 5′UTR (untranslated region) [The upstream activating sequences in n factor . UASs ar region) ,18. UAS  region) .The TATA box is a hexamer  cThe location and activity of the transcription start sites are constrained by the position of the TATA boxes . Indeed,CYC1 constitutive promoter (pCYC1—throughout the whole paper we will refer to promoters by using the letter “p” in front of the name of the gene they precede in the yeast genome), which leads the expression of iso-1-Cytochrome C, has been studied deeply and is now well characterized [Among the natural promoters in budding yeast, the cterized ,28,29,30cterized .CYC1 gene transcription in glucose-containing media, whereas in the presence of lactate, UAS1 and UAS2 contributes almost equally to CYC1 expression [pCYC1 contains two UASs, three TATA boxes, and at least six TSSs ,29,30. Upression .TATA boxes start at position −106, −52, and −22. They activate, respectively, 3, 4, and 2 transcriptions start sites among the six that have been reported so far see .S. cerevisiae, hybrid promoters are built by merging DNA elements—from distinct natural constitutive and inducible promoters—that play diverse roles in transcription. A traditional way to construct hybrid promoters requires combining the upstream activating sequence(s) of one promoter with the core sequence of a different promoter [CYC1 promoter UASs with a 365-nt-long sequence from the GAL10 gene . The same hybrid promoter was used, a few years later, for studying yeast fermentation [S. cerevisiae [In the yeast promoter . This tepromoter ,33 localentation  and exprrevisiae .S. cerevisiae. Furthermore, by fusing the upstream activating sequences of the widely employed galactose-inducible GAL1 promoter to the CYC1 and LEUM core promoters (with the addition of other short DNA motifs) they made a library of galactose-inducible hybrid promoters that spanned an about 50-fold dynamic range in gene expression  whose accumulation provokes toxicity effects into yeast cells [PGK1 promoter with the GAL1,10 intergenic region. It is worth mentioning that, in this work, pPGK1 was turned into a testosterone-responsive promoter as well after replacing its UAS with androgen-responsive elements  promotestress conditions such as a temperature increase (heat-shock [Other hybrid promoters are induced by particular cellular at-shock ), oxidatat-shock , variatiat-shock , and lacat-shock .Hybrid promoters have been, mainly, engineered by combining UASs (or other binding sites for yeast-endogenous activators) and core promoters. Alternatively, synthetic promoters have been constructed by changing the sequence of native yeast promoters via mutations, deletions, nucleosome removal, and intron insertion see .ADH1 promoter proved to enhance gene expression [TEF1 promoter was generated via error-prone PCR [S. cerevisiae promoters . Error-prone PCR was adopted also to generate a variant of the pheromone responsive FUS1 promoter, termed pFUS1J2, that showed almost undetectable basal expression and higher gene expression upon induction. pFUS1J2 found application in a positive feedback loop [Back in the nineties of the last century, shortened variants of the constitutive pression ,50. Laterone PCR ,53. The rone PCR  to make ack loop  and an Rack loop .HIS3 promoter with the insertion of the Gcn4-binding sequence flanked by two poly(dA:dT) sequences  that highly disfavored nucleosome formation. Moreover, Curran and co-authors [GPD promoter, and binding sites for glycolytic TFs) joint by random DNA sequences. By applying the NuPoP algorithm to these synthetic sequences, promoters with higher strengths (up to 20-fold than the corresponding template) were computed. These new synthetic promoters had minimal homology with those in the S. cerevisiae genome [Nucleosome removal is a strategy to increase promoter activity. According to it, Raveh-Sadka et al.  built a -authors  improved-authors . Then, me genome .Another, different way to affect promoter activity demands the insertion of intron sequences along the 5′UTR, as illustrated in . This teA different strategy to design synthetic inducible promoters was illustrated in  and requ5NYAWNN(Arich)6). They were separated by 30 nt only. UASs, whose length was 10 nt, were placed upstream of the TATA box. Between them, a 30-nt-long spacer randomly generated, was inserted. By using three UASs, a core promoter, and the 30-nt-long spacer in between, a synthetic promoter almost as strong as pGPD but only 116-nt long (versus the 655 nt of pGPD) was built  site [DEG1 terminator [CYC1 terminator.Novel synthetic constitutive core promoters, of moderate strength, were also generated by placing the −16…+71 region of pCYC1 downstream of either natural or synthetic minators . The seq(A) site . The eff(A) site . Thereforminator , the weaorthogonal to their chassis i.e., they should not interfere with the molecular biology of the host cell. Therefore, to have orthogonality, the transcription factors expressed by a circuit should not bind DNA outside of the synthetic gene network. This is achieved, in S. cerevisiae, by using bacterial proteins as TFs and inserting their operators within the sequences of the circuit promoters that need to be repressed or activated (see Transcription factors (repressors and activators) are proteins that wire together two or more TUs inside a genetic circuit. They regulate the transcription initiation rate of their target promoters upon binding specific operators. Synthetic gene circuits should be ated see A,B.In the following, we describe yeast synthetic promoters regulated by eight different kinds of bacterial proteins. Importantly, each protein can be controlled by an environmental signal such that gene expression is induced or inhibited by either adding/removing chemicals to/from the cell solution or growing cells under particular conditions.E. coli repressor protein that binds a rather long operator  organized in two subunits separated by a single base pair [AD) and worked as an activator for the two engineered promoters. More yeast synthetic activators were, later on, constructed by fusing either the GAL4 DNA binding domain (GAL4DBD) or the whole LexA to activating sequences from E. coli such as B42 [LexA is an ase pair ,67. The ase pair  turned pase pair , they plh as B42  and the h as B42 . In parth as B42 .S. cerevisiae and could be adopted to control transcription via molecules of beta-estradiol. They made a chimeric activator by fusing together the GAL4DBD, the HBD(hER), and the VP16 activation domain from the Herpes simplex virus [ante litteram. A synthetic promoter was engineered by replacing the UAS of pGAL1 with eight copies of the lex operator. In this way, the promoter was activated by a chimeric protein made of LexA fused to the aryl hydrocarbon receptor (AHR), which contains an activation domain. Like HBD(hER), AHR interacts with Hsp90 in the absence of its ligand(s). Only in the presence of alpha- or beta-naphthoflavone, LexA-AHL was able to translocate into the nucleus and stimulate the production of the LacZ reporter protein.In 1992, LexA served as a DBD for a chimeric repressor that contained also the chromatin remodeling factor Ssn6 . One yeaex virus . In the ex virus . A planaex virus  in what S. cerevisiae. In this work, the minimal CYC1 promoter was preceded by eight lex operators bound by LexA-VP64 .A similar structure for a synthetic promoter was adopted in  for the S. cerevisiae genes. Fluorescence, from each promoter, was measured at low concentrations of beta-estradiol (up to 100 nM). The core pGAL1 showed both the highest response  and—together with the core pSPO13—the best inducibility (on/off ratio).Synthetic promoters based on a minimal pCYC1 (where only the TATA box at position −52 was kept) extended with a variable number of lexO (1 to 8) were used in  as targeENO1 promoter on the antisense strand and the core PGK1 promoter on the sense strand.Rantasalo and co-authors  describeS. cerevisiae and filamentous fungi (Trichoderma resei and Aspergillus niger) [universal gene-expression system for fungi that was validated in six yeast species and the two filamentous fungi mentioned above [In a following work by the same group, more bacterial proteins were tested both as activators and repressors of engineered core promoters from s niger) . SrpR, Ps niger) ). The sys niger) —to builded above .tet-off. In a later work, Gossen and co-authors [reverse version of TetR (indicated as TetR’) that binds the DNA only in the presence of tetracycline. As a consequence, the tetracycline—TetR’-VP16 system was termed tet-on.The bacterial repressor protein TetR, in its wild-type configuration, binds the DNA at a 19-nt-long sequence, the tet operator—tetO. Antibiotics belonging to the family of tetracycline dock to TetR and induce a change in its structure that prevents any further DNA binding . Gossen -authors  construcCYC1 promoter. Gene expression was enhanced by fusing one or two copies of VP16 to TetR. Moreover, the insertion of a linker between TetR and VP16  was a simple way to further increase the potency of tet-off-based systems. In a later work from the same lab, Belli’ and co-authors [S. cerevisiae repression moieties, Ssn6 and Tup1, the former providing a tighter transcriptional control. In a more recent work [S. cerevisiae. Remarkably, Mnaimneh et al. [S. cerevisiae via promoters down-regulated via a cassette of seven tet operators.Garí et al.  realized-authors  gave a ment work , the proent work  were redh et al.  analyzedS. cerevisiae was characterized in [GAL1 promoter. The repression level due to TetR increased with the operator number. Moreover, higher repression was achieved by placing the operators closer to the TATA box rather than to the TSS. One of the promoters containing two tetO sites was the basis for the construction of a library of 20 more repressed promoters that covered a wide range of gene expression and inhibition levels. This was achieved by mutating randomly the region around the two tet operators [The repressive action of the bare TetR on gene expression in rized in . Seven sperators . These nTATA-less promoter engineering was given in [S. cerevisiae PFY1 promoter. pPFY1 displays, instead of the TATA-box, a poly-T sequence that provokes a DNA bend probably responsible for the access of RNA polymerase II to the core promoter. The new tetracycline-controlled promoter (pPFY1i) showed very low basal fluorescence expression in the absence of tetracycline and reached around 75% of pPFY1 fluorescent level upon induction with tetracycline. Both pPFY1 and pPFY1i were used as targets for TALORs  binds three operators with different affinities. An ideal, 20-nucleotide-long operator (lacO) sequence has been proposed by Oehler and co-authors [In -authors . LacI acS. cerevisiae was engineered by Grilly and co-authors [ADH1 promoter with the insertion of a single lacO downstream of the TATA box. Two copies of this synthetic promoter, termed pADH1-i, were employed to drive, in the presence of IPTG, the synthesis of the bacterial proteins ClpX and ClpP that dimerize to form the ClpXP protease. In this circuit, the green fluorescent protein gene, responsible for the production of an output signal, was fused to the ssrA tag that is recognized by ClpXP. Therefore, in the absence of IPTG, the network expressed green fluorescence, whereas, in the presence of IPTG, ClpXP degraded the tagged GFP with a consequent decrease in fluorescence.The first LacI-repressed synthetic promoter in -authors  that modGAL1-based LacI-repressed promoters. They employed these promoters as components of genetic timers. Moreover, they built a synthetic promoter hosting tetOs in tandem together with a single lacO. The promoter, highly derepressed only in the presence of both tetracycline and IPTG, was a part of an I1 FFL.Ellis and co-authors  construcCYC1 promoter (containing the TSSs) with a portion of the VPH1 promoter (enclosing the TATA box). The two promoter pieces were joined by a short spacer where one or two binding sites for bacterial proteins ) were placed to achieve repression of transcription. Single-input YES and NOT gates together with two-input AND, N-IMPLY, and OR gates were generated by changing the number and the relative position of the operators, beside the copy (integration) number of the plasmid containing the bipartite promoter.Similar promoter configurations, based on simultaneous repression by TetR and LacI, were engineered in  to mimicCYC1 promoter was modified with the insertion of a lac operator downstream and five binding sites of the human androgen receptor upstream of the TATA box at position −52. The human androgen receptor binds the DNA only in the presence of testosterone and works as an activator. In the absence of IPTG, LacI binds the corresponding operator and prevents fluorescence expression. If cells are induced only with IPTG, the fluorescence level stays low. Thus, only in the presence of both chemicals, the promoter expresses the green fluorescence protein in high quantity—mimicking, in this way, an AND gate. A different dual-mode promoter has been implemented [A dual-mode promoter, i.e., a promoter that can be both activated and repressed, was engineered by Mazumder and McMillen . A weak lemented  and complemented  by usingXylose is a sugar abundant in lignocellulosic lysates and used, by microbes, in the production of several chemicals . Xylose-S. cerevisiae, XylR from three different bacteria. Each XylR targeted a synthetic promoter made of the UAS from pTEF1 and the core GAL1 promoter was modified with a xyl operator (xylO) downstream of the TATA box. Since the sequence of xylO is not conserved among bacterial species, a different hybrid promoter was constructed for each of the three XylRs. By following an analogous strategy, Wang and co-authors [GPM1 promoter that kept its own UAS. Three promoters contained a single xylO located 19 nucleotides downstream or 1 nucleotide upstream of the TATA box, or 1 nucleotide downstream of the UAS. In the fourth promoter, two xylOs were employed: one downstream of the UAS, the other upstream of the TATA box. Furthermore, after deriving a consensus sequence for xylO, a library of synthetic promoters was realized through PCR with primers containing degenerate operator sequences. The new operators were located upstream of the TATA box.Teo and Chang  cloned, -authors  built xyTEF promoter of the filamentous fungus Ashbya gossypii. The original promoter sequence was modified with the removal of one of the two TATA boxes and the addition of one or two xyl operators in the proximity of the only TATA box left. XylR was fused to the yeast chromatin remodeling protein Ssn6 to enhance the repression of transcription. The best performance in terms of induction of lacZ expression—up to 25-fold—was obtained when the TATA box was flanked, at each side, by a xyl operator.Hector and Mertens  construcreverse XylR from E. coli (EcXylRR) that binds the DNA only in the presence of xylose. EcXylRR was fused to the strong composite VPRH  activation domain. The original biosensor was implemented in the yeast Yarrowia lipolytica. Here, four synthetic promoters were built by placing a xylO upstream of the TATA box of the complete, strong TEF promoter, and of three core promoters obtained from the constitutive TEF, hp4d, and ACC1 promoters. The synthetic promoter based on the TEF core promoter was the most performant in terms of on-to-off ratio with a 20.9-fold gain upon xylose induction. This biosensor configuration was then ported to S. cerevisiae cells. With respect to the background fluorescence, the circuit returned a 43.3-fold higher signal in the presence of xylose.A different xylose biosensor was engineered in  by exploE. coli and Vibrio cholerae) were realized in [GAL1 promoter. Strong fluorescence repression was achieved with the E. coli FadR binding a 3-operator-containing pGAL1. Such a biosensor can be adopted to regulate protein expression—and, thus, balance the cellular metabolism—according to the availability of fatty acid intermediates inside yeast cells. In a later work by the same lab, Teo and Chang [tandem operators of the E. coli FadR along the core promoter sequence and replacing the original UAS with that of either constitutive  or inducible (CUP1 and PHO5) yeast promoters. The first three new synthetic promoters were used to detect fatty acids in media containing glucose, whereas the last two promoters permitted to realize Boolean gates responding to fatty acids and either the presence of copper (AND gate) or the absence of phosphates (N-IMPLY gate).Fatty acid (and fatty acyl-CoA) sensing devices based on the FadR repressor from two different bacterial sources  that binds a 34-nt-long operator (fapO) only in the absence of malonyl-CoA. A synthetic promoter repressed by FapR was built on the yeast GPM1 promoter. Only one UAS was kept along the promoter sequence and either 1 or 2 fapO sites were placed upstream of the TATA box. The best biosensor configuration (lowest leakage and highest dynamic range) was achieved with fapR gene on an episomal plasmid (high protein number) and a single fapO in the engineered pGPM1 (4.17-fold gain). David and co-authors [Malonyl-CoA is a basic building block for the biosynthesis of value-added compounds such as fatty acids, polyketides, and flavonoids. Li and co-authors  engineer-authors  construcE. coli FadR repressor. FadR operators replaced fapO sites on the synthetic pTEF1 promoters in [RTC3, GGA2, and LPP1) were shown to lead to an about 80% increase in the level of fatty alcohols.A similar pTEF1-based synthetic promoter was employed in  to impleoters in . This biTEF1 promoter re-engineered with three fapO sites upstream of the TATA box conserved the strength of the native TEF1 promoter and was 5.4-fold repressed by FapR fused to the Mig1 repression domain.The work in  was contLEU2 promoter modified with a fapO site 57 nt upstream of the TATA box. This biosensor gave a 53.5-fold increase in fluorescence expression in the absence of the malonyl-CoA and an 82% decrease in the presence of 20 uM cerulenin . Upon replacement of fapO with either a FadR or a XylR binding site in the synthetic promoter, the circuit was turned into a biosensor for either fatty acid or xylose.A novel design for malonyl-CoA, fatty acid, and xylose biosensors was presented in . These bE. coli repressor protein MetJ is enabled to adhere to the DNA at its operator sequence, metO.SAM (S-adenosylmethionine) is a methyl donor for the modification of multiple molecules such as DNA, RNA, and proteins. Upon binding SAM, the S. cerevisiae by, employing a novel synthetic activator, MetJ-B42, targeting a synthetic CYC1 promoter hosting metO upstream of the TATA box (metO-pCYC1) [A SAM biosensor was engineered, in O-pCYC1) . metO-pCAcinetobacter sp ADP1, which responds to CCM . In bacteria, BenM is an activator that binds the DNA independently of its ligand. In the presence of CCM, BenM induces a structural change in the DNA that enhances the binding of RNA polymerase to the promoter. Initially, the author constructed three synthetic promoters based on the yeast CYC1 promoter. They placed a single BenM operator (benO) upstream of the TATA box at position −106 (T1 variant) or upstream of the TATA box at position −52 (T2 variant). A third configuration (T1-T2) contained both benOs. When BenM was produced under pTEF1, fluorescence increased in T1 (20 folds) and T1–T2 (5 folds) promoter configuration proving that BenM works, as an activator, also in yeast without the need for any AD. By growing the cells in a 1.4 mM CCM solution, however, only a modest enhancement in fluorescence expression was detected. Therefore, the whole system had to be optimized to return a clear response to CCM. To this aim, the best promoter design turned out to be the “209bp_CYC1_BenO_T1”. This synthetic promoter was used to build three more biosensors by replacing benO with the operator of other LTTRs, namely: FdeR (sensing naringenin); ArgP (L-arginine), and MdcR .Skjoedt and co-authors  built biS. cerevisiae demands to modify yeast promoters with the insertion of operators that, usually, reduce promoter activity when placed within the core sequence. To avoid this issue, zinc finger proteins (ZFPs), TAL effectors (TALEs), and CRISPR-Cas have been adopted to engineer new, orthogonal TFs. These three systems allow targeting almost every sequence within a native promoter by customizing a DNA-binding domain (ZFPs and TALEs) or expressing a short RNA molecule (CRISPR-Cas).The use of bacterial proteins as orthogonal TFs in A zinc finger domain (ZFD) is a structural motif, made of about 30 amino acids, that recognizes and binds a DNA triplet. Adjacent ZFDs constitute the DBD of zinc finger proteins. Synthetic TFs have been constructed by fusing activation or repression domains to either a naturally occurring DBD from zinc finger proteins or a novel DBD obtained by joining from three to six ZFDs. ZF-based DBDs proved to bind their target DNA sequence with high specificity .CYC1 promoter sequence was turned into a synthetic activated promoter by placing, upstream of the TATA box, a variable number of either the binding site of the natural murine zinc-finger protein Zif268 [A minimal n Zif268  or shortn Zif268 .de novo by merging 4 ZFDs (Z4). Z3 and Z4 were built to overcome the problem of the high number of off-target binding sites (over 500) associated with GEV. Z3 had 11 predicted binding sites along the S. cerevisiae genome, whereas Z4 was designed to bind only its own operator. Both Z3 and Z4 activated a synthetic promoter made by replacing the 3 UASs of the GAL1 promoter with 6 of their binding sites. Z3 was further characterized in a later work by the same group [McIsaac and co-authors  modifiedme group  where itme group  to buildbase-specifying residue [Transcription activator-like effectors (TALEs) are, like ZFPs, a family of proteins characterized by a modular DNA-binding domain . TALEs,  residue . The DBDCYC1 promoter, a variable number of TALEs’ operators (up to 16) both in forward and reverse sense . The promoters targeted by TALE-VP64 were bound also by dCas9-VP64-based activators (see below). TALEs outperformed dCas9-VP64. The best TALE-activated promoter showed a 400-fold enhancement, in fluorescence, upon binding of the TAL effectors. The activation efficiency scaled with the operator number and, interestingly, was higher with reverse operators. Fine-tuning was possible by inserting one or two mismatches in the operators, whereas three mismatches, in general, prevented TALEs from binding the DNA.Besides the TALOR-repressed promoters  previousStreptococcus pyogenes).CRISPR –Cas (CRISPR-associated protein)  represenspacers alternated with direct repeats, is transcribed into a long pre-CRISPR RNA sequence (pre-crRNA). The spacers are short pieces of DNA that were inserted into the bacterial chromosome after previous infections. The pre-crRNA is processed by the joint action of a further short RNA molecule (named tracrRNA—transactivating crRNA), RNAse III, and SpCas9. As a result, the complex SpCas9-crRNA:tracrRNA is released. The spacers (included in the crRNA) that are complementary to the intruder DNA bind it by base pairing in the proximity of the protospacer adjacent motifs  and allow SpCas9 to induce a double-strand break in the foreign DNA, which provokes its fast degradation [ad hoc.Upon intrusion of foreign DNA, a CRISPR array, made of radation . Jinek eradation  showed tradation  of usageradation   of a chosen 20-nucleotide-long target sequence (termed a1) upstream of the TATA box. As expected, fluorescence expression increased with a1 number, though without a remarkable difference between 3 and 12 a1 sequences. The configuration with six a1 sites was utilized into an AND gate where dSpCas9-VP64 expression was conditioned to the presence of galactose in the cell culture and the sgRNA transcription was triggered by tetracycline. Similarly, Machens and co-authors [CYC1 promoter preceded by a variable number (from 2 up to 16) of arbitrarily chosen binding sites (BSs). Fluorescence expression, however, did not scale with the operator number and was, usually, very low for promoter configurations with 16 BSs. The short distance between adjacent BSs (6 nucleotides instead of the 20 in [Farzadfard and co-authors  studied -authors  measuredhe 20 in ) was indS. cerevisiae. scRNAs are sgRNA extended, for instance, with one or two MS2 hairpins where the MCP protein, fused to a repression or an activation domain, can bind.The tet operator was the target for dSpCas9 in . Seven tCYC1 promoter preceded by a UAS from pGPD and modified in the proximity of the TATA box with two target sites complementary to as many sgRNAs. Inputs for the NOR gate were two distinct sgRNAs that formed a complex with dSpCas9-Mxi1 . The our domain . NOR gatS. cerevisiae promoters and the usage of bacterial proteins as orthogonal transcription factors in yeast gave important results in the eighties and nineties of the last century. Techniques and methods developed in those years have been inherited by the Synthetic Biology community and are still currently used in the construction of many synthetic gene circuits. Over the years, more bacterial proteins were found to be working in yeast and were used to build, principally, biosensors whenever they interacted with chemicals of various origins or reacted to stress conditions. The real novelty of the last decade was in TF, rather than promoter, engineering with the discovery of TAL effectors and, especially, the CRISPR-Cas systems.The engineering of hybrid Synthetic Biology, however, aims at the rational, model-driven design of both circuits and DNA components—such as promoters. Mathematical instruments, from System Biology ,136, hav


Drosophila melanogaster, the Piwi-piRNA pathway plays a key role in the transcriptional silencing of TEs considered to be exerted mostly through the establishment of H3K9me3 histone marks recruiting Heterochromatin Protein 1a (HP1a). Here, using RNA-seq, we investigated the expression of TEs and the adjacent genomic regions upon Piwi and HP1a germline knockdowns sharing a similar genetic background. We found that the depletion of Piwi and HP1a led to the derepression of only partially overlapping TE sets. Several TEs were silenced predominantly by HP1a, whereas the upregulation of some other TEs was more pronounced upon Piwi knockdown and, surprisingly, was diminished upon a Piwi/HP1a double-knockdown. We revealed that HP1a loss influenced the expression of thousands of protein-coding genes mostly not adjacent to TE insertions and, in particular, downregulated a putative transcriptional factor required for TE activation. Nevertheless, our results indicate that Piwi and HP1a cooperatively exert repressive effects on the transcription of euchromatic loci flanking the insertions of some Piwi-regulated TEs. We suggest that this mechanism controls the silencing of a small set of TE-adjacent tissue-specific genes, preventing their inappropriate expression in ovaries.Insertions of transposable elements (TEs) in eukaryotic genomes are usually associated with repressive chromatin, which spreads to neighbouring genomic sequences. In ovaries of  Drosophila melanogaster, HP1a, also known as Su(var)205, defines large pericentromeric and telomeric heterochromatin domains but also interacts with many sites in euchromatin, including insertions of transposable elements (TEs) and protein-coding genes =UAS-Dcr-2.D}1, w [1118]/+; UAS-Piwi-RNAi(#101658 VDRC)/P{w[+mC]=GAL4-nos.NGT}40; UAS-HP1a-RNAi/+. The identified TE insertions sites in this genome were then used for a transcriptomic analysis of the four studied genotypes . To detect TE insertion sites in the line with germline knockdowns of HP1a and Piwi, we sequenced the genome of double-GKD females with the genotype http://www.fruitfly.org/p_disrupt/TE.html, accessed on 13 December 2021) and the annotated TE insertion sites in the FlyBase (r.5.57) reference genome using the pipeline PoPoolationTE2 [To perform whole-genome sequencing, genomic DNA was isolated from 25 whole female flies according to the standard procedure . The paiationTE2 . Overallp < 0.05 and, in some cases, TPM fold change >2. Heterochromatic genes were defined as genes residing on chromosomes 4, U, U extra, chr2RHet, chr2LHet, chr3RHet, chr3LHet and chrXHet, as well as residing in heterochromatin on chromosome arms 2R, 2L, 3R, 3L and X, according to the cytological borders of heterochromatin listed in Reference [For RNA isolation, approximately 20–50 ovaries from 0- to 1-day-old Ctrl, Piwi-GKD, HP1a-GKD and double-GKD females were manually dissected in cold PBS. RNA was isolated using Trizol reagent , yielding over 2 µg for each genotype, in two biological replicates. rRNA was depleted using the Illumina Ribo-Zero rRNA Removal Kit . The cDNA library was prepared with random primers, and single-end deep sequencing was carried out on the Illumina HiSeq 2000 in the Laboratory of Evolutionary Genomics, Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University . 15.1–18.5 million reads (after leaving out the rRNA reads) were obtained per library. The 50-nt reads were aligned to dm3/R5 genome and transcriptome assembly via STAR , countedeference . RNA-seqWe visualised the transcript levels (log2 RPM) derived from the +/− 3-kb genomic regions flanking individual TE copies as heat maps centred at the TE insertion sites. For this, we used the genomation package for R . For eacTo prepare the metaplots centred at the TE insertions, normalised RPM values around the TE insertions were obtained as follows. The genome was split into 500-nt bins, RPM values for the control and three knockdowns in the bin containing the insertion , as well as 6 adjacent bins in each direction, were taken and lined up according to the insertion orientation (+ or − strand). In order to compare transcription activation with the control, we normalised all the values onto the RPM value in the central bin in the control. If that value was below 1, we added 1 to every RPM value to avoid giving too much credit to TEs with low RPM values around them. Then, all the resulting values were averaged for every TE family or for all the TE insertions.Boxplots with ratios of log2 TPM values of genes residing either in the neighbouring 3 kb or 10 kb of a TE insertion in knockdowns relative to the control were plotted for each TE family that had at least 10 genes in the vicinity of its insertions.For visualisation of the sequencing tracks, the IGV genome browser was used.U test was used either for comparisons of two sample distributions or for comparing the median of distributions with zero. The RPM on the TE-adjacent sequences were averaged over all the insertions and compared between the knockdowns and control. For the analysis of the upregulation of genes adjacent to the TEs, we used one-tailed M–W tests for the log2 ratios of the TPM values upon knockdowns to the control compared with zero as the median. To assess whether the activation of genes residing near TE insertions is indeed attributed to such localisation, we used the permutation (non-random) test. First, we calculated the p-value of the activation of genes that were near TEs via the M–W U test of their ratio of log2 TPM values in the knockdowns to log2 TPM values in the control for each TE family. Then, we took the same number of random genes and assessed their activation in a similar manner 10,000 times. The resulting p-value was the number of times the p-value of random gene activation was less than the p-value of actual gene activation divided by 10,000.The Mann–Whitney (M–W)


Clostridium thermocellum is a thermophilic bacterium recognized for its natural ability to effectively deconstruct cellulosic biomass. While there is a large body of studies on the genetic engineering of this bacterium and its physiology to-date, there is limited knowledge in the transcriptional regulation in this organism and thermophilic bacteria in general. The study herein is the first report of a large-scale application of DNA-affinity purification sequencing (DAP-seq) to transcription factors (TFs) from a bacterium. We applied DAP-seq to > 90 TFs in C. thermocellum and detected genome-wide binding sites for 11 of them. We then compiled and aligned DNA binding sequences from these TFs to deduce the primary DNA-binding sequence motifs for each TF. These binding motifs are further validated with electrophoretic mobility shift assay (EMSA) and are used to identify individual TFs’ regulatory targets in C. thermocellum. Our results led to the discovery of novel, uncharacterized TFs as well as homologues of previously studied TFs including RexA-, LexA-, and LacI-type TFs. We then used these data to reconstruct gene regulatory networks for the 11 TFs individually, which resulted in a global network encompassing the TFs with some interconnections. As gene regulation governs and constrains how bacteria behave, our findings shed light on the roles of TFs delineated by their regulons, and potentially provides a means to enable rational, advanced genetic engineering of C. thermocellum and other organisms alike toward a desired phenotype. Clostridium thermocellum   enzymes . The bac enzymes , cellulo enzymes , genetic enzymes  genome e enzymes , computa enzymes , and beyC. thermocellum reflects the general interest in using this thermophilic bacterium to convert biomass to biofuels  have been characterized for their roles of regulating cellulolytic enzymes in response to sugar molecules, particularly GlyR3  facilitates high-throughput selection of TF-DNA complexes. DNA fragments released from heat-denatured TFs are then barcoded and amplified for multiplexed next-generation sequencing. The advantages of using IVTT and an in vitro environment for TF-DNA binding come with the costs of potentially producing DNA-binding-incompetent TFs due to protein misfolding or the absence of the essential cofactors or effector molecules necessary to induce a DNA-binding TF conformation. DAP-seq was first developed using Arabidopsis TFs , experimental validation, and prior knowledge of the TFs’ homologues along with their regulatory targets to reconstruct a gene regulatory network in C. thermocellum. This network encompasses TFs and respective regulons (sub-networks) with previously characterized homologues (such as LexA Clo1313_1449 and Rex Clo1313_2471) as well as those not previously known. In this reconstructed gene network, there are regulons as small as a two-gene operon and as large as nearly 100 genes in 83 operons. To our knowledge, this study is the first large scale DAP-seq analysis of bacterial TFs and adds substantially to the body of work describing transcriptional regulations in Clostridium thermocellum.In this study, we screened for genome-wide DNA binding sites for nearly one hundred Acetivibrio thermocellus and Ruminiclostridium thermocellum also known as Hungateiclostridium thermocellum which is commonly known and referred to herein as Clostridium thermocellum DSM 1313  Joint Genome Institute Integrated Microbial Genomes and Microbiomes  and following the protocol for gram-positive bacteria. The gDNA concentration was measured with the Qubit 3.0 Fluorometer . A total of 33 μg of gDNA was sent to the University of Idaho Genomics Resources Core for the preparation of a DAP-seq-ready DNA library. At the facility, the low molecular weight fragments of 50–100 bp were removed using a DNA bead purification protocol . The gDNA was then sheared to ∼200 base pair fragments using a S-series focused ultrasonicator . Primer adapters were ligated to the ends of the 200 bp fragments generated by the sonication step. The efficiency of the ligation steps of the adapters to our gDNA fragments was measured using quantitative PCR  indicating that approximately 25% by weight of the gDNA fragments were successfully ligated at both ends, yielding ∼8 μg of adapter-ligated DNA.Total genomic DNA (gDNA) was extracted from fresh overnight cultures of AATGATACGGCGACCACCGAG ATCTACACNNNNNNNNACACTCTTTCCCTACACGAC-3′; and i7_duplex_primer_1, CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGT. The “NNNNNNNN” region of these primers represent the unique DNA indices (barcodes), the underlined sequence represents the p5 (i5) and p7 (i7) Illumina sequences.The qPCR reaction was performed using primers that contain the Illumina adapter sequence (p5 and p7) as well as a unique index sequence (barcode) designed for multiplexing the sequencing reaction step. An example of these primers is: i5_duplex_primer_1, 5′-E. coli and cloned by GENEWIZ into pFC20A  at the SgfI and XhoI cut sites. The final constructs encode TFs with a C-terminal HaloTag. We performed DAP-seq assays following published protocols  was used for IVTT of putative TFs according to the manufacturer’s recommendation. For a large-scale assay of 131 putative TFs in triplicates, a total reaction volume of 150 μl and 3 μg of the plasmid DNA template was initially setup and subsequently split into three DAP-seq reactions. The IVTT reactions were performed anaerobically at 37°C for 4 h. The TF-HaloTag fusions were loaded onto Magne® HaloTag® Beads (Promega) while nutating for 1 h at room temperature under anaerobic conditions. During this time, TF proteins with HaloTags formed covalent attachments to the magnetic beads. The TF-bound beads were then washed with PBS+NP40 solution three times to discard untagged, non-specific proteins and resuspended in 120 μl of PBS+NP40. The magnetic beads with covalently bound TFs were split into three wells in a 96-well PCR plate. We also performed small-scale DAP-seq assays involving few targeted TFs and conditions. Five technical replicas were setup for each TF in the small-scale DAP-seq and reaction volumes were proportionally scaled for five parallel replicates. A negative control plasmid without a TF was also constructed in such a way that the TF amino acid sequence was replaced with the amino acid sequence MHHH, resulting in a construct that encodes a MHHH-HaloTag fusion. Whether the experiment is setup to be small- or large-scale, an IVTT without expressing a TF is performed to provide a negative control for downstream assays.Genes encoding putative TFs were codon optimized for expression in rotocols  with a fTF-binding gDNA fragments were pulled-down by DAP-seq assays. We added 40 μL of Qiagen elution buffer containing 12.5 ng of gDNA library to TFs that were previously attached to magnetic beads. The TFs and DNA were incubated at 55°C for 2 h under anaerobic conditions while nutating. Free DNA was separated from bead-bound TF-DNA complexes by repeatedly washing the magnetic beads with fresh PBS+NP40. TF-bound DNA was recovered for analysis by denaturing the TFs at 98°C for 10 min. PCR was then performed to simultaneously amplify the adapter-ligated DNA and add barcoded adapters to facilitate multiplexed sequencing as previously described . Five miTwo sets of DAP-seq experiments were performed in addition to the large-scale assay of 131 putative TFs. Eight TFs were re-assayed in the absence and presence of various amounts of the soluble fraction of cell lysate to test if co-factors in the lysate would improve TF-DNA binding and therefore detection of binding sites by DAP-seq (because the TFs may change their protein conformation upon binding to an effector). Finally, 13 TFs were assayed in five parallel replicates to assess reproducibility of the assay, and a new DNA library was freshly prepared for this set of DAP-seq experiment. Each assay included the MHHH-HaloTag construct negative control in addition to the TFs.1 and were of overall high quality (above Q-30). Reads were mapped to the Clostridium thermocellum DSM 1313 genome (RefSeq Accession NC_017304) using bowtie2  were performed to confirm TF-DNA binding sequence motifs identified by DAP-seq data analyses. The C-terminal HaloTag coding sequence was replaced with a 6xHis tag by amplifying the TF-encoding pFC20A plasmids with Long-Amp Taq DNA polymerase using primers prSDH_051 (AGTGATGGTGATGGTGATGGGATCCATCG TTATCGCTCTGAAAGTA) and prSDH_052 (TGGATC CCATCACCATCACCATCACTAATAGAATTCTAGAGTCGAC CTGCAGG). These primers introduce KpnI cut sites allowing the PCR-linearized vector to be closed by restriction digest and ligation. The accuracy of the DNA sequences in these constructs were validated by commercial Sanger sequencing (GENEWIZ).E. coli BL21 DE3 cells. One-liter batches of LB supplemented with ampicillin (100 μg/ml) were inoculated with 10 mL of overnight cultures. Protein production was induced with 1 mL of 0.5 M IPTG when the culture reached an optical density of approximately 0.8. Induced protein production continued overnight at room temperature. Cells were harvested by centrifugation, resuspended in ∼30 mL of PBS with 25 mM imidazole and lysed on ice using a QSonica Q500 sonicator with a CL-334 probe for 5 min on a 50% duty cycle at 30% power. Cell debris was pelleted by centrifugation at 27,000 relative centrifugal units (×g) for 55 min at 4°C. The supernatant was incubated with 1 mL of pre-equilibrated HisPur nickel-NTA resin  in a rocking ice-water bath for 30 min. The resin was collected by passing all the supernatant through the accompanying spin column with repeated centrifugation at 600 × g for 2 min. The resin was washed and spun five times with 5 mL of PBS containing 25 mM imidazole. Proteins were eluted by washing three times with 1 mL of PBS containing 250 mM imidazole. The eluent was pooled and concentrated using a 0.5 mL, 10 kDa-cut-off Amicon concentrator and buffer-exchanged with PBS containing NP40. Total protein concentration was measured using BioRad protein quantification assay with BSA as the standard. The proteins were kept at 4°C or on ice to reduce precipitation.His-tagged TFs were expressed in p-value) site in the MEME-ChIP aligned peak sequences. The probe sequences include a representative sequence of the motif plus 10 bp on each flank in context of the peak from which it was derived. Probes without matches to the expected motif (p > 10–3) were used as negative controls. After combining the probe and protein, the volume was brought to 10 μL using PBS+NP40, and 3 μL of 5x sucrose-based loading buffer was added. Samples were loaded onto 10-well, 6% polyacrylamide DNA retardation gels  and subjected to 100 V for 1 h with room temperature 0.5× TBE  as the running buffer without temperature control. Gels were stained in 50 mL of running buffer with 1× for 20 min while rocking and imaged on a BioRad GelDoc. EMSA images were quantified using ImageJ (EMSAs were performed with 0.5 μM double-stranded DNA oligo probes (IDT DNA) and various amounts of semi-pure TFs. Probes were synthesized to match the best  were used to estimate the probability that a given peak sequence belongs to the set of sequences encompassed in a position-specific scoring matrix (PSSM) with a threshold of p < 10–4 . The PSSp < 10–5) or a DAP-seq peak, along with the corresponding region from similar genes in related organisms, were scanned for conserved binding sites using RSATs footprint-scan function .4 Default GOMo parameters were used except negative controls were derived from 10,000 sets of shuffled scores.The biological role of a TF is often characterized by the collective functions of its target genes. We tested if gene functions were enriched among genes with high scoring matches to the motif using GOMo . Such anNetworks of TFs and their putative target genes were visualized using Cytoscape 3.8.0 . Edges cClostridium thermocellum. The core progression of our approach is depicted in Control over gene transcription is one of several layers of regulations that govern essential biological functions. We used a combination of complementary experimental and bioinformatic techniques to characterize TFs from n = 3 for each TF). Sequencing of multiplexed DAP-seq libraries yielded approximately 350 million paired-end, 150 bp-long reads, resulting in an overall genome sequencing depth of about 75X. The preferential binding of TFs resulted in an overabundance of reads at specific loci, or DAP-seq peaks. Ten TFs yielded peaks ranging from 1.1 to 12.2 times the local depth of mapped reads relative to the negative control, and the TFs that resulted in TF-DNA binding events are summarized in Q-value), the nearest ORF and the peak’s relative position to the ORF is in We initially identified and assayed 131 putative TFs (listed in n = 5 for each condition). The cell lysate is presumed to provide cellular components including cofactors, metabolites, and other proteins which may be required to trigger TF-DNA binding. In these assays, we found peaks for a TF  for which no peaks were identified in the previous DAP-seq assay. However, similar counts of peaks were identified whether lysate or water was added. Consistent to the first DAP-seq experiment, a few peaks were found for Rex_2471, but the addition of lysate had no appreciable affect (data not shown). These peaks for Rex were not a significant addition to the 128 previously identified peaks. Peaks for both proteins are listed in In a later DAP-seq experiment, we selected nine potentially cofactor- or effector-dependent TFs  and repeTo test the reproducibility of the resulting peaks, we repeated the DAP-seq assay (with freshly prepped DNA library and proteins) for the 10 TFs for which peaks were found in the original screen but with five replicates each. The relative sequencing depth at the best peak remained within range of the original assay, 13.8 vs. 12.2. These peaks are also listed in In all, peaks were identified for 11 TFs, 10 during the large-scale assay. After the assays were performed, we discovered that 40 of the 131 putative TFs assayed did not have a recognizable DNA binding domain or are expected to bind DNA without sequence specificity based on the domains detected by the conserved domain search at NCBI , 2017. TArabidopsis transcription factors. In the Arabidopsis study, bZip- and NAC-family proteins showed cross-experiment correlations with Pearson coefficients between 0.71 and 0.99  MEME-ChIP motif in the collective set of peaks or an equivalent motif derived from a single experiment that shows higher-order symmetry, such as an inverted repeat. The only exception is the motif reported for Xre_2225 (A sequence logo depicting a representative motif for each TF is shown in Xre_2225 . The motq < 0.05) with the published DNA binding motifs from TFs with similar functions. The motif for Rex _2471 is also similar to the motif of RexA from Clostridium acetobutylicium though it is not present in these databases (q < 0.5), except for BlaI_1845 and Xre_2225. These thresholds are excessively inclusive, and therefore these two motifs appear to be unique to this study.We compared these motifs to those available in the MEME-Suite databases for prokaryotes . The motatabases . These fin vitro. Our EMSAs demonstrate sequence-dependent TF-DNA binding in favor of probes with their respective predicted motif for six of the 11 TFs . This requirement compensates for the cross-experimental variability of peaks in our DAP-seq experiments. Half of all peaks discovered in the high throughput screen contained the binding motif of their respective TF. This ratio improved to about three quarters in our repeat experiments. The use of five parallel replicates and lower throughput/multiplexing are factors that may contribute to this improvement. While we used motif-scanning to filter for relevant peaks, others have used RNA-seq to corroborate their findings from DAP-seq . These PSSMs specify the likelihood of finding a particular nucleotide at each position in the motif sequence and can be used to identify TF binding sites. We set a requirement that all peaks in a regulon must have a TF binding site as identified by a significant match to the PSSM  were found among genes with high scores for the LexA_1449, Rex_2471, TetR_0692, and GntR_1482 motifs. A single gene ontology term for SOS response to DNA damage was found for LexA_1449. The Rex_2471 motif was upstream of genes enriched in ontology terms for redox activity, glycolysis, and transcription regulation. The other motifs each identified transposition and nucleic acid binding as conserved target functions. Transposition was additionally associated with the TetR_0692 motif. Because individual binding sites are not identified by GOMo, we did not attempt to infer individual target genes from this search. However, these functional themes generally agree with our phylogenetic footprinting experiments and the predicted functions of these TFs.Our searches for conservation of specific functions using GOMo were subject to the same limitations and had similar performance as the footprint-scan from RSAT. Conserved functions , are of particular interest for their involvement in regulating central carbon metabolism. LacI TFs alter between DNA binding and non-binding conformations in response to the absence or presence of a specific ligand, usually a sugar molecule. All three C. thermocellum LacI proteins were tested in our large-scale screen but no DAP-seq peaks were detected under this condition. We were, however, able to identify peaks for GlyR2 from a targeted DAP-seq experiment.The three LacI repressor-type TFs encoded in glyR2 deletion strain  that was upregulated in the glyR2 mutant  in a n strain . Direct 313_1398 . The gen2 mutant . This evman5A (clo1313_1398), where binding and regulation had been previously observed, to be a false negative DAP-seq result. However, DNA binding motifs upstream of man5A and other potential false negatives were recaptured by the bioinformatic motif searches. The best match to the GlyR2 PSSM . The other three cellulosome components with GlyR2 binding sites are glycoside hydrolases family 5 (clo1313_0413), a xylanase (clo1313_2530), and a lipolytic enzyme (clo1313_1783).We consider the absence of a peak upstream of yR2 PSSM  in the k2 motifs  and in t2 motifs . BecauseC. thermocellum.These findings are consistent with the functional role of GlyR2 as a mannobiose-responsive transcription repressor . We haveIn the case of the GlyR2 regulon, our bioinformatics results played the role of expanding the regulon to include genes that were missing from the DAP-seq data. For the next three regulons that we discuss, our bioinformatics results play a nearly opposite role. The sequence scanning results for the two BlaI TFs  and Xre_2225 provided evidence to explain why so few DAP-seq peaks were within non-coding sequences. In one case these results also provide justification for eliminating a gene identified by DAP-seq from the regulon.BlaI_0696 and BlaI_1845 belong to the penicillinase/beta-lactamase repressor (BlaI) family of TFs. BlaI TFs often regulate their own operon and operons containing beta-lactam resistance genes by binding as a dimeric complex to inverted repeat DNA sequences . Only twin vivo. In fact, this evidence also suggests that these BlaI TFs target genes with highly ordered binding sites for four protomers . These peaks each contained two matches to the BlaI_0696 representative motif , transduction (blaR1 and blaI), and response functions (peptidoglycan synthesis) apparently sufficient to limit the effects of beta-lactams. The clo1313_0696 operon consists of genes encoding BlaI_0696, two BlaR1s, and a hypothetical protein (Clo1313_2238). Since all other genes in this regulon are involved in sensory and signal transduction functions, we suspect that clo1313_2238 encodes the antibiotic response mechanism.The functions of the genes in the BlaI_1845 and BlaI_0696 regulons are consistent with a response to beta-lactams. Beta-lactam antibiotics achieve bactericidal activity by irreversibly inhibiting peptidoglycan synthesis enzymes. The resulting inability to restructure the cell wall is fatal for the bacteria. Cells avoid beta-lactam toxicity by deploying either beta-lactamase enzymes or alternative cell wall synthesis enzymes. Beta-lactams are sensed by BlaR1 proteins. In the presence of beta-lactams, BlaR1 proteolytically cleaves BlaI, thus relieving inhibition of the BlaI regulon . The sinclo1313_2226-clo1313_2225 operon, encoding a Type II toxin-antitoxin pair, respectively , and both of these high scoring sites lie upstream of clo1313_2226 . Other predicted binding sites and DAP-seq peaks are upstream of genes with roles in genome methylation and maintenance, genes encoding components of the ribosome, and a large operon encoding cell wall synthesis enzymes involved in vancomycin resistance.Consistent with the established knowledge of TetRs, TetR_0692 binding sites  and DAP-clo1313_1690-1688). Two binding sites occur at the apices of these peaks forming a pseudo-palindromic sequence TGCAAATcATTTGCA  from E. coli binds to and represses transcription of a zinc-specific ABC transporter and other genes in the zinc-bound state  and the Type II toxin-antitoxin operon mentioned earlier (clo1313_2226-clo1313_2225). Coupling transcription of a mechanosensitive ion channel to zinc transport may help maintain osmotic balance. The control of a Type II TA system by a zinc uptake regulator could provide a mechanism for cells to enter a non-growth state until there is a change in their environment. However, an additional layer of transcriptional repression control on the TA system may provide a more compelling benefit than zinc deficiency-coordinated expression.Other putative gene targets worth noting include a mechanosensitive ion channel . The results for the three TFs discussed in this section do not seem to converge and coalesce to the same extent.clo1313_1279, clo1313_0692, and a two-component transcriptional regulator clo1313_2868) and a few genes involved in DNA replication . The GntR_1482 regulon also contains clo1313_1372, a peptidase M24, and clo1313_1170, a encoding hypothetical protein. The peptidase gene was the only gene to be identified as a GntR_1482 target in both DAP-seq experiments, and both of these genes have two putative binding sites in their upstream regions. The rest of the regulon is comprised of genes of various predicted functions and can be found in The GntR_1482 and Xre_0026 motifs both feature elements of dyad symmetry commonly found in TF binding sites  does notclo1313_0222 and clo1313_0223 gene products via a programmed translational frame shift. However, the AraC_0222 DAP-seq results were the most consistent of all samples . The correlation between the Xre_0026 motif and sporulation genes is not quantitative, and these genes are a small portion of the predicted target genes. The full list is available in The predicted Xre_0026 regulon includes several genes related to sporulation  for LexA_1449 were found in upstream non-coding regions in both experiments. The DNA binding sequence motif derived from all DAP-seq peaks matches the symmetry . We wereclo1313_1449), as was also the case for B. subtilis LexA . Fourteen target genes were also predicted to be part of a conserved regulon using footprint-scan from RSAT (C. bescii Rex operon (clo1313_1885- clo1313_1881), a putative hydrogenase system transcriptional regulator (clo1313_1572) and Rex_2471 itself (p < 10–5) and footprint-scan, demonstrating the complementarity between these two approaches. The fact that an interaction between Rex and this hydrogenase regulator was previously observed in the literature  to the characterized Rex proteins from Clostridium acetobutylicum  was downregulated under furfural-induced stress while Cthe_1798 (homolog of Rex_2471) had no detectable change , LexA _1449, Xre_2225, and Rex _2471. Negative self-regulation minimizes the basal expression of a TF and reduces noise in transcription . It seemClostridium thermocellum. To our knowledge, this is also the first report of large-scale, DAP-seq screening of bacterial TFs. This systems biology approach expedites the discovery of multiple TFs’ DNA binding sequence motifs and subsequently their regulatory targets, thereby revealing regulatory information not previously known. Our bioinformatic searches served to both remove false positive peaks and identify novel and anticipated members of the regulon that were missing from the DAP-seq data. This highlights the complementarity of the DAP-seq and bioinformatic analyses. Coupling with traditional EMSA to validate DAP-seq and bioinformatic findings, we established a framework to transform large sets of data from DAP-seq to physiologically relevant information. Overall, the TFs characterized herein and their respective regulons provide a valuable foundation for future scientific and engineering endeavors.This work is a substantial addition to the body of knowledge pertaining to transcription factors and gene regulatory networks in PRJNA723898. An interactive Cytoscape file containing the complete gene regulatory networks and supporting evidence summarized in this work will be made available through the NREL Data Catalog, accessible at https://data.nrel.gov/submissions/161. The NREL Data Catalog is the electronic catalog for data generated by federally funded research at the National Renewable Energy Laboratory.The datasets presented in this study can be found in online repositories. DAP-seq raw sequencing reads that support the findings of this study have been deposited in the Sequence Read Archive with the BioProject ID JM, AG, Y-PC, and KC conceived and planned DAP-seq experiments which were carried out by JM and Y-PC. AG performed quality control analysis and interpretation of sequencing reads. SH performed motif discovery and subsequent bioinformatics analysis and performed EMSAs. SH and AG led initial drafting of the manuscript. KC secured funding to enable this work. All authors contributed to finalizing the manuscript.The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The anchorage of Ras isoforms in the membrane and their nanocluster formations have been studied extensively, including their detailed interactions, sizes, preferred membrane environments, chemistry, and geometry. However, the staggering challenge of their epigenetics and chromatin accessibility in distinct cell states and types, which we propose is a major factor determining their specific expression, still awaits unraveling. Ras isoforms are distinguished by their C-terminal hypervariable region (HVR) which acts in intracellular transport, regulation, and membrane anchorage. Here, we review some isoform-specific activities at the plasma membrane from a structural dynamic standpoint. Inspired by physics and chemistry, we recognize that understanding functional specificity requires insight into how biomolecules can organize themselves in different cellular environments. Within this framework, we suggest that isoform-specific expression may largely be controlled by the chromatin density and physical compaction, which allow (or curb) access to “chromatinized DNA.” Genes are preferentially expressed in tissues: proteins expressed in pancreatic cells may not be equally expressed in lung cells. It is the rule—not an exception, and it can be at least partly understood in terms of chromatin organization and accessibility state. Genes are expressed when they can be sufficiently exposed to the transcription machinery, and they are less so when they are persistently buried in dense chromatin. Notably, chromatin accessibility can similarly determine expression of drug resistance genes. They predominate in different cancer types and the distributions of the mutations differ as well . The covalent palmitoylation thioester modification linkages are reversible; the farnesyl thioether linkages are not. The HVR of KRas4B can also be phosphorylated. The introduction of the large, negatively charged phosphoryl group dissociates KRas4B from the negatively charged plasma membrane  is disordered, precluding crystallization. However, its populated conformations have been sampled by NMR and molecular dynamics (MD) simulations  roles, localization, sublocalization, and more. To avoid re-reviewing the topics which were covered, we refer the reader to these recent excellent publications. These include subcellular localization and considering exploiting the membrane in therapeutics  of chromosome 11 at position 5, the KRAS gene is located on the p arm of chromosome 12 at position 12.1, and the NRAS gene is on chromosome 1 at position 13.2. Even though these are positions along the linear chromosome sequence organization, and to date data about their 3D locations are unavailable, the distinct locations of isoforms suggest distinct organization and expression patterns.Most chromatin conformation capture experiments focused on intrachromosomal interactions. Recent observations reveal that the patterns of interchromosomal interactions are tissue-specific, differing between the heart and liver  interacts with KRas4B  CaM–KRas4B-specific binding reduces the number of available free CaM molecules for CaEven though there are some sequence differences in the catalytic domains, the distinction among Ras isoforms rests mainly in their HVR membrane-binding segments Fig. . This diRAS isoform-specific gene scale topologically associating domains (TAD) will be identified not only in specific tissues (Szabo et al. All proteins are encoded and can be expressed by all cells (Kosti et al. G12C (Zeng et al. To date, pharmacology has successfully targeted KRas4B


DnaA has been shown to regulate genes involved in a variety of cellular events including those that trigger sporulation, DNA repair, and cell cycle regulation. DnaA’s dual functions (replication initiator and transcription factor) is a potential mechanism for DnaA to temporally coordinate diverse cellular events with the onset of chromosome replication. This strategy of using chromosome replication initiator proteins as regulators of gene expression has also been observed in archaea and eukaryotes. In this mini review, we focus on our current understanding of DnaA’s transcriptional activity in various bacterial species.In bacteria, DnaA is the most conserved DNA replication initiator protein. DnaA is a DNA binding protein that is part of the AAA+ ATPase family. In addition to initiating chromosome replication, DnaA can also function as a transcription factor either as an activator or repressor. The first gene identified to be regulated by DnaA at the transcriptional levels was  The twoaA boxes . This fuaA boxes . In thisaA boxes . DnaA haaA boxes . The spednaA mutants of Escherichia coli revealed that the copy number of ori decreased, while DnaA levels increased simultaneously at nonpermissive temperatures, thus accurately predicting that DnaA had a positive involvement in the onset of chromosome replication and a negative involvement in regulating its own synthesis  were shown to decrease the activity of the dnaA promoter, and second, decreased DnaA levels able to bind the dnaA promoter (by adding dnaA binding sites on a plasmid that titrated DnaA levels away) were shown to increase the activity of the dnaA promoter , and QseB , repression of dnaA expression was not observed in P. putida. Interestingly, the overexpression of dnaA (only the open reading frame) from E. coli under the same broad host range plasmid construct in P. putida does result in repression of dnaA’s transcription  or bound to ADP (Kd 100 nM)  can be modulated using an ATP-dependent molecular switch. In  100 nM) . The pro E. coli . The cel E. coli . As a tr E. coli . DnaA–AT binding .E. coli. Ribonucleotide reductase (RNR) encoded by nrdAB catalyzes the reduction of ribonucleotides to deoxyribonucleotides  has been proposed to activate the transcription of a set of three essential genes encoding proteins involved in cell cycle regulation: FtsZ , MipZ (inhibitor of FtsZ polymerization), and GcrA  (R357A resulted in C. crescentus cells over-initiating replication (E. coli (DnaAR334A) was shown to bind ATP but was unable to hydrolyze ATP  . The explication . The corlyze ATP . In C. cand gcrA . In B. sin vitro , suggestC. crescentus and B. subtilis utilized innovative approaches to separate DnaA’s transcriptional activity from its function as a replication initiator  were shown in vitro to have promoter regions with affinity for purified His6DnaA. Notably, one of the DnaA-dependent genes identified was the gene encoding for GcrA, the global regulator of Caulobacter cell cycle  knocked out. The expressions of dnaA and dnaN were engineered at different loci regulated by two different inducible promoters. Using this system, 91% of 339 total number of genes that displayed DnaA-dependent transcriptional regulation were shown to be indirectly regulated by DnaA via Sda. DnaA regulates the transcription of sda by directly binding at sda’s promoter region . Second, in C. crescentus, DnaA regulates the levels of the active form of the replication initiator (DnaA–ATP) by regulating the expression of hdaA  has been shown in E. coli and proposed in C. crescentus  commonly found in the same operon downstream of dnaA  and dnaB (DNA helicase), both of which were identified as part of the DnaA transcriptional regulon by delaying dnaA transcription (DnaA as a transcription factor regulates the levels of substrates for DNA synthesis and of components of the replisome (molecular machinery required for chromosome replication). In ynthesis . The rolescentus . In E. c of dnaA . In C. ccription .polA (E. coli involved in DNA replication and DNA repair (uvrB (component of Nucleotide Excision Repair NER system) and recN [involved in repair of double-stranded breaks  initially suggested DnaA’s involvement in ftsZ expression; however, the three DnaA boxes were later shown not to play a role in ftsZ transcription  in vivo using chromatin immunoprecipitation assays  encoding the flagellar transcriptional regulator FlhD  by binding the promoter region during exponential growth and high nutrient availability  production  composed of six subunits . In humaresponse . The abiresponse .oris flanking the dnaA gene (as in B. subtilis and Helicobacter pylori; dnaA. How are these two different functions of DnaA coordinated and differentiated over the cell cycle? Most of the work done on DnaA’s nucleotide switch has been performed in E. coli. How widespread is DnaA’s nucleotide switch used to regulate DnaA’s transcriptional activity in other bacterial species? To conclude, the transcriptional activity of DnaA has only been characterized in a few bacterial species. Within these few, the specifics of DnaA’s transcriptional regulon vary significantly. Thus, the characterization of replication initiators’ roles in coordinating various cellular events will continue to provide exciting results for years to come.In this mini review, several aspects of the transcriptional activity of DnaA were discussed including autoregulation, its mechanism of regulation, and its role in chromosome replication and other key cellular events. Many exciting questions about the transcriptional activity of DnaA remain to be answered. For instance, DnaA has been shown to act as an activator and a repressor of transcription . Is therAll authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Epigenetic changes induced in the early developmental stages by the surrounding environment can have not only short-term but also long-term consequences throughout life. This concept constitutes the “Developmental Origins of Health and Disease” (DOHaD) hypothesis and encompasses the possibility of controlling livestock health and diseases by epigenetic regulation during early development. As a preliminary step for examining changes of epigenetic modifications in early embryos and their long-lasting effects in fully differentiated somatic tissues, we aimed to obtain high-throughput genome-wide histone H3 lysine 4 trimethylation (H3K4me3) profiles of bovine blastocysts and to compare these data with those from adult somatic tissues in order to extract common and typical features between these tissues in terms of H3K4me3 modifications. Bovine blastocysts were produced in vitro and subjected to chromatin immunoprecipitation-sequencing analysis of H3K4me3. Comparative analysis of the blastocyst-derived H3K4me3 profile with publicly available data from adult liver and muscle tissues revealed that the blastocyst profile could be used as a “sieve” to extract somatic tissue-specific modifications in genes closely related to tissue-specific functions. Furthermore, principal component analysis of the level of common modifications between blastocysts and somatic tissues in meat production-related and imprinted genes well characterized inter- and intra-tissue differences. The results of this study produced a referential genome-wide H3K4me3 profile of bovine blastocysts within the limits of their in vitro source and revealed its common and typical features in relation to the profiles of adult tissues. During preimplantation development, dynamic epigenetic rearrangements occur, including substantial changes in DNA methylation and histone modifications, which regulate specific and heritable patterns of gene expression4. As the epigenome is dynamically formed during the preimplantation period, environmental intervention-induced changes in epigenome formation during this period have been considered as one of the possible causes of the long-lasting influences induced by the periconceptional environment1. This concept constitutes the “Developmental Origins of Health and Disease” (DOHaD) hypothesis and encompasses the possibility of controlling health and diseases in later life by epigenetic regulation during early development.The periconceptional period of mammalian embryonic development is a critical window during which diverse environmental factors surrounding the embryo have not only short-term consequences such as effects on immediate embryonic development, but also long-term consequences including lasting influences on metabolic, developmental, and etiologic processes throughout gestation and even during postnatal and adult life7. For example, in vitro handling of ruminant early embryos in assisted reproductive technology increases the risk of fetal overgrowth syndrome9, whereas maternal nutrition, stress, or illness during pregnancy can affect productive traits such as postnatal growth, milk yield, carcass composition, and fertility7.Some phenotypic changes in the field of livestock production can be discussed in the context of DOHaD given that early life events during prenatal and early postnatal development often affect traits, including those of economic importance11. As a preliminary step for examining the changes of epigenetic modifications in early embryos and their long-lasting effects in later life, the elucidation of the common and typical features of epigenetic modifications between these two developmental stages is worth studying. In particular, accumulating evidence suggests that histone modifications at developmentally important genes, compared with DNA methylation, are more susceptible to the surrounding environment during the preimplantation period13. In the present study, we aimed to obtain genome-wide profiles of histone H3 lysine 4 trimethylation (H3K4me3), a representative marker of active chromatin, in bovine blastocysts produced in vitro and to compare these profiles with those of adult liver and muscle tissues, which have been deposited in public databases. We examined whether we can extract the common and typical H3K4me3 features of early embryos and adult somatic tissues.Although the epigenetic modifications that occur during the early developmental period, which persist in differentiated tissues, are considered a major mechanism of DOHaD, there is few comparative studies of histone modifications between early embryos and fully differentiated somatic tissuesGAPDH, which is a representative positive region for H3K4me3 modifications14. Figure 15 -seq analysis of H3K4me3 using three groups of bovine blastocysts (n =  ~ 11 per replicate) derived from two independent IVF procedures. Pairwise comparisons of the ChIP signals in the triplicates showed the high reproducibility of our method  were designated as the liver peaks. On the other hand, the 20,298 overlapping peaks identified from our ChIP data from blastocysts  were designated as the blastocyst peaks. Then, we merged these peak groups and extracted 1899 and 7901 liver- and blastocyst-specific peaks, respectively  compared with liver (1.02 ± 0.62), whereas ALDH5A1 exhibited the opposite pattern (0.71 ± 0.23 for blastocysts and − 1.07 ± 0.14 for liver). The genes largely contributing to PC2 exhibited relatively large intra-tissue deviations; for example, PPP3CA exhibited − 0.21 ± 1.01 in blastocysts and 0.32 ± 1.25 in liver , the liver data were relatively comparable with our blastocyst data in terms of available read numbers, average profiles of the gene groups of interest Figs. a, and th8 and genomic imprinting is also closely associated with the meat production traits of livestock23. The TSSs of 105 known imprinted genes listed in a previous bovine study8 and two differentially methylated regions (DMRs) related to bovine fetal overgrowth (KvDMR1 of KCNQ1OT1 and IGF2R/AIRN DMR) were selected for investigation, and we found that 30 TSSs and the two DMRs had H3K4me3 peaks both in blastocysts and liver. The average profile plot of the 30 TSSs again exhibited overall similar profiles between the two different tissues  and KCNQ1OT1 (with KvDMR1), both of which are implicated in fetal overgrowth syndrome8, exhibited a relatively high contribution to intra-tissue differences (PC2). In addition, we further investigated the correlation between the levels of H3K4me3 around TSSs and the expression levels of these imprinted genes calculated from publicly available RNA-seq data. Despite the independent experiments, the levels of H3K4me3 in both tissues exhibited significant correlations with the gene expression levels  to elucidate common and typical epigenetic modifications between early embryos and differentiated somatic tissues. Interestingly, the H3K4me3 peaks of blastocysts could be used like a “sieve” to extract somatic tissue-specific peaks in genes closely related to tissue-specific functions. Although the genes with the top 100 highest peak occupancy rates in blastocysts, liver, and muscle did not represent the specific functions of each tissue, their counterparts after subtracting genes with common peaks  clearly did Figs. . These rALDH5A1 and CROT). On the other hand, PC2 might mirror intra-tissue differences rather than inter-tissue ones, for example, such as PPP3CA  such as for fat score  and beef marbling score (35.2% for Japanese Black bulls22). Although DNA methylation has been characterized as a major code of genomic imprinting, recent studies reported DNA methylation-independent imprinting that is controlled by histone methylation26. In addition, the global dysregulation of imprinted genes in assisted reproductive technology-induced fetal overgrowth is often independent of DNA methylome epimutations27. These findings suggest the importance of histone methylation in the expression of imprinted genes. In the present study, we categorized H3K4me3 modifications on imprinted genes into tissue-specific , suggesting that these H3K4me3 modifications are common in early embryos and hepatic tissues and diverse among individuals and/or given conditions. Interestingly, within the limits of comparing among independent (unrelated) experiments, we could observe the significant correlations between the H3K4me3 levels of these imprinted genes and their expression levels in both blastocysts and liver  and was carried out in accordance with the Regulation on Animal Experimentation at Kyoto University. The bovine ovaries used in the study were purchased from a commercial abattoir as by-products of meat processing, and the frozen bull semen used for in vitro fertilization (IVF) was also commercially available. In vitro production of bovine embryos by IVF was performed as previously describedg and the supernatant (200 μL) was transferred to a new tube. The 200-µL sample of sheared chromatin was divided into a 10-µL aliquot as “input” and the rest (190 µL). The latter aliquot was processed for ChIP using 3 µg anti-H3K4me3 antibody  as indicated in the user manual. The input and ChIPed DNA was decrosslinked, purified, and resuspended in 40-µL low-EDTA TE buffer. The DNA samples were processed for library preparation for next-generation sequencing by using a Next Gen DNA Library Kit (Active Motif) following the manufacturer’s manual. The specific enrichment of H3K4me3 in the ChIP-seq libraries was validated by quantitative PCR for positive (1st exon–intron boundary of GAPDH) and negative (2nd exon of MB) regions.ChIP for small cell numbers was performed with a Low Cell ChIP-Seq Kit (Active Motif) according to the manufacturer’s manual (version A3) with some modifications. The blastocysts were freed from the zona pellucida by using pronase before crosslinking with formaldehyde. After crosslink-quenching, the sample was sonicated to shear chromatin using a Bioruptor UCD-250 (Cosmo Bio) for 30 × 30 s with 30-s pauses in ice-water. The sample was centrifuged for 2 min at 18,000 × 33. The mapping duplicates were removed by Picard (http://broadinstitute.github.io/picard/). The peaks were called in the ChIP samples relative to the respective input samples using MACS34. The annotation of called peaks to genomic regions was generated using CEAS35 and the peak occupancy rates were in its output. Average H3K4me3 enrichment profiles and heat maps were generated by ngs.plot21, and peak areas were calculated from its output. Gene ontology analysis was performed using the DAVID tool18. ChIP-peaks were visualized using the Integrative Genomics Viewer (IGV)36. The publicly available raw data for bovine liver and muscle were processed as described above except for the lack of input sample in the muscle sample. The common and specific peaks between samples were identified using bedtools (https://bedtools.readthedocs.io/en/latest/) with the default and − v option, respectively. PCA with autoscaling of H3K4me3 peak areas and Pearson correlation analysis with average H3K4me3 peak areas and merged RNA-seq data were performed using SPSS (SPSS Inc.).Sequencing was performed on a HiSeq2500 (Illumina) as sigle-end 51-base reads. The sequencing reads were quality checked and aligned to the bovine genome  except for scaffolds using Bowtie16; ChIP-seq of bovine muscle (longissimus dorsi), GSM1517452 of GSE6193619; RNA-seq of bovine blastocysts, GSM1265773, GSM1265774, and GSM1265775 of GSE5241537; and RNA-seq of bovine liver, GSM4799566 and GSM4799567 of GSE158430 (one of the split files of pair-end reads were used). For RNA-seq data, the three (blastocyst) and two (liver) datasets were merged and expression levels in RPKM values were calculated as previously described38. The genes were evenly divided into three categories as high, medium, and low expression levels according to the calculated RPKM values.The following raw data from publicly available databases were used: ChIP-seq of bovine liver, Bull4 and Bull5 of E-MTAB-2633Supplementary Information 1.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.


TNP1 gene is abundantly expressed in the regulation of spermatogenesis, and its sequence is remarkably well conserved among mammals. Genomic analysis, by sequencing and computational approach, was used to identify the novel polymorphisms and to evaluate the molecular regulation of TNP1 gene expression in Sahiwal cattle breeding bulls. DNA samples were sequenced to identify novel single nucleotide polymorphisms (SNPs) in the TNP1 gene. Modern computational tools were used to predict putative transcription factor binding in the TNP1 promoter and CpG islands in the TNP1 promoter region. In the TNP1 gene, four SNPs, three TATA boxes, and one CAAT box were identified. One CAAT box was discovered at 89 bp upstream of start site ATG. The computational analyses indicated that the polymorphisms inside the promoter sequence results in an added HNF-1 transcription factor binding site. In contrast, the other variations may remove the naturally occurring SRF transcription factor binding site. The CpG islands in the TNP1 promoter region were predicted to be absent by the MethPrimer program before and after SNP site mutations. These findings pave the way for more research into the TNP1 gene's promoter activity and the links between these SNPs and reproductive attributes in the Sahiwal breeding bulls.Transition nuclear proteins (TNPs), the principal proteins identified in the condensing spermatids chromatin, have been found to play a key role in histone displacement and chromatin condensation during mammalian spermatogenesis. One such gene belonging to the TNP family called  TNP1 gene in Bos taurus is found on chromosome 2q42-q43 [https://www.ncbi.nlm.nih.gov/protein/XP019841056.1) with approximately 20% lysine and 20% arginine spread equally [TNP1 is an extensively produced protein during spermatogenesis regulation, and its sequence is substantially similar among mammals [TNP1 gene appear to be linked to DNA degradation in individuals with azoospermia and varicocele [Bulls have gained major importance in animal breeding since a specific bull can mate numerous females. A lack of breeding competence in the bull can have a greater influence on the productivity of a herd than female reproductive issues. Bull genomics is considered the primary material through which genetic improvements can efficiently be accomplished. Some in fertility and subfertility problems have been observed for the culling of Murrah, Sahiwal, and KF bulls. One of the significant causes of infertility and subfertility is spermatozoa maturation . Anomali2q42-q43  and encoTNP1 and TNP2 renders the mouse model sterile, whereas subfertile null mice mutants lack TNP1 and/or TNP2 [TNP1 gene at locations 205, 340, and 346 bp, and all these SNPs are located in the intronic regions. When the influence of this polymorphism on mass activity, individual motility, and spermatozoa maturation was investigated, it was found to have a substantial impact on spermatozoa development but was nonsignificant on mass activity and sperm motility [Similarly, the lack of both /or TNP2 –13. Moremotility –16.TNP1 gene in breeding bulls were identified using a DNA sequencing technique. Furthermore, computational tools were utilized for promotor prediction, prediction of transcription factor (TF) binding sites and CpG islands, and the influence of discovered SNPs on the CpG Islands. The primary purpose of the present study includes creating a foundation to confirm the effect of SNPs on TNP1 gene expression and the relationship between reduced spermatogenesis in breeding bulls and the SNPs.In current study, SNPs of the n = 50) of Sahiwal cattle breeding bulls were collected from Semen Production Unit (SPU), Qadirabad, Sahiwal, Pakistan. Semen samples of bulls were transported in liquid nitrogen and stored temporarily in the freezer at −20°C before DNA extraction.Semen straw samples (260/OD280 ratio of 1.83 ± 0.005 were used for further work.Extraction and purification of genomic DNA from semen straws were conducted using the phenol-chloroform method described . Agarosehttp://frodo.wi.mit.edu/cgi-bin/primer3/primer3http://www.slow.cgi) of the TNP1 bovine gene (NCBI Reference Sequence: NC_032651.1) , 2.5 μL 10x PCR buffer with 2.5 μL MgCl2, 1 μL of each forward and reverse primer (both at 10 mol/L), 2.5 μL of dNTPs, 0.5 μL of 5U of Taq DNA polymerase, and 13 μL of nuclease-free water (Thermo Fisher Scientific). The PCR amplification program was as follows: initial denaturation step at 94°C for 3 minutes, followed by 35 cycles of denaturation step at 94°C for 30 seconds, annealing step at 58°C for primer set for 30 seconds, and extension at 72°C for 1 minute, and final extension step at 72°C for 10 minutes. To store PCR products, −20°C refrigeration temperature was used.Primer3 software was used to create gene-specific oligonucleotide primers  at 4°C. The supernatant was discarded, and pellets were washed with 100 μL of 70% ethanol. Then, pellets were dissolved in 15 μL of deionized water. After sequencing PCR, precipitated PCR products were sequenced using dye-labeled dideoxy terminator sequencing using ABI Genetic Analyzer 3130 XL . The sequencing results of the samples were aligned by BLAST and ClustalW with the TNP1 reference gene sequence of Bos indicus (NCBI reference sequence: NC_032651.1). The background noise in the amplified PCR product sequence chromatogram was trimmed using 4Peaks software (https://nucleobytes.com/) from both sides and pairwise alignment was obtained with NCBI ref. seq. accession. No. NC_032651.1 using NCBI online BLAST software. MEGA6 [TNP1 gene amplified from breeding bulls.Gel electrophoresis was used to evaluate the quality and size of amplified PCR products on 1.2 percent agarose gel. Ethanol was used for the precipitation of PCR products. 40 e. MEGA6 , 19 was TNP1 gene, including promoter region, CpG islands, and transcription factor binding sites, were predicted and analyzed through different computational tools.Different functional elements of TNP1, three distinct computational tools were used in this investigation, including Promoter Scan [The promoter is the central DNA element separated into distal, proximal, and core regions and is meant to regulate the transcription of genes. To infer the promoter areas of ter Scan , Promoteter Scan , 22, andter Scan . PromoteTNP1 promoter's binding sites of the transcription factor in breeding bulls. AliBaba2.1 server is for context-specific identification of transcription factor binding sites, and prediction is based on constructing matrices on the fly from TRANSFAC 4.0 sites [A computational algorithm called AliBaba2.1  was used.0 sites .http://itsa.ucsf.edu/∼urolab/methprimer) was utilized to predict the CpG islands present in the TNP1 gene's promoter.CpG islands are those regions in DNA where GC content is high. They are prominently found within promoter region or in the vicinity of promoter region which act as markers for the regulation of gene expression when undergoing methylation. MethPrimer is a computer program based on Primer3, and it is specifically developed for designing primers for methylation mapping. It works by taking the DNA sequence as input and then searching the sequence for potential CpG islands . In the All allelic and genotypic frequencies for newly found SNPs were quantified and tested in the current investigation using the earlier developed method .TNP1 belongs to a class of TNP genes important in histone-to-protamine replacement during spermatid nuclear transformation. This gene family is believed to alter sperm nuclear chromatin to ensure stability after transformation to a DNA-protamine complex [TNP1 gene expression causes failure of spermatogenesis to occur, resulting in a round spermatid arrest [TNP1 gene have been shown to impair chromatin structure of sperms, ultimately leading to diminished fertility in mice [TNP1 gene in breeding bulls affect their reproductive abilities is still lacking. Moreover, there are no studies on the TNP1 gene SNPs on the local breeding bull population in Pakistan. So, in the current study, the SNPs in the promotor region of TNP1 gene, which can primarily lead to reduced spermatogenesis in the Sahiwal breeding bulls  is investigated. complex . Suppresd arrest . Moreove in mice , as well in mice  and the  in mice . A broadTNP1 promotor region and disagreement with them concerning the position of TATA boxes [We found four SNPs in the promoter region at −568 nt, −468 nt, −446 nt, and −288 nt from transcription start site upstream, while positions of these SNPs in BLAST pairwise alignment were 75C > T, 161T >C, 183G > A, and 341T > C . Three TTA boxes . We idenTA boxes .TNP1 gene via changes in transcription factors.The sites where DNA sequences are with transcription factors are called binding sites. Alterations in transcription factor binding sites could cause a significant impact on transcription factor binding to gene regulatory areas and gene expression products . We examTNP1 promoter region. As per the findings, a C ⟶ T polymorphism happened in the gene's promoter region, culminating in an extra binding site for the HNF-1 transcription factor. Another polymorphism, i.e., T ⟶ C at the locus may remove the initial binding site of SRF transcription. The G ⟶ A variation can also result in the loss of RAP transcription factor binding sites, while the T ⟶ C polymorphism could remove the original C/EBPalp binding site. As a result, such mutations, especially at recently found loci, can potentially eliminate the original transcription factor binding sites while generating new transcription factor binding sites, changing the control of TNP1 expression in cattle.RNA polymerase recognizes and binds DNA at the promoter region. DNA sequence changes in this area may alter binding sites of the transcription factor to influence gene expression. CpG islands are unmethylated gene sequences that can be coupled with particular transcription factors to enable regular expression . When CpTNP1 promoter area before and after SNP site mutations, we used MethPrimer software. This was done to investigate the influence of such SNPs on modifications of the anticipated CpG island in the promoter region of TNP1. The MethPrimer software  projected no CpG islands in the TNP1 gene promoter area  breeding bulls was examined. Genomic sequencing analyses of the gene and its various important sites identified four SNPs, three TATA boxes, and one CAAT 89 bp upstream of the ATG start codon. The identified SNP and variations in other genetic elements are expected to be correlated with altered spermatozoa maturation. In the TNP1 promoter region, a SNP resulted in an added HNF-1 transcription factor binding site, which can alter the TNP1 expression controlling genetic apparatus. Moreover, the absence of CpG islands in the promoter region of TNP1 was also observed, which is expected for transcription of the TNP1 gene significantly. The present study's findings could help us understand the role of various genetic sequences and how their variations affect the expression of TNP1 gene in Sahiwal breeding bulls. However, the limited size of the sample population is a hindrance to forming a definite conclusion. So, in the future, a larger sample size is required to establish a causal role of the TNP1 gene control on reproductive attributes of breeding bulls, the predictions made in the study form the groundwork for future research.In the present investigation, the effect of SNPs on