Insertion of an insertion sequence (IS) element in the regulatory region upstream of the bgl promoter activates expression of the operon. The repression mechanism involves the histone-like nucleoid structuring (H-NS) protein with two DNA binding sites, one in the region upstream of the promoter, and the other within the first structural gene of the operon, bglG. The detailed mechanism of repression is not well understood. Here, we first show two terminators flanking bglG are not required for bgl operon silencing. Instead, several lines of experimental evidence clearly suggest that the silencing mechanism involves looping of the DNA between H-NS’s two DNA binding sites. H-NS is known to preferentially bind to AT-rich curved DNA, and such regions are found in the vicinity of both sites. We show that strong repression is abolished by (1) preventing H-NS self-oligomerization while retaining DNA binding, (2) preventing or reducing H-NS binding to the bgl operon regulatory region, and (3) preventing or reducing H-NS binding to the binding site in the bglG gene. We also show that the phase of the DNA between these two binding sites is not important, and that large insertions of DNA in the putative loop region do not diminish repression. These results imply that H-NS depends on DNA looping to exert strong repression.The  In spite of this, the bgl operon can be activated by mutations, and it then becomes inducible in the presence of β-glucosides, so that E. coli cells can utilize β-glucosides for growth, leading to the Bgl positive (Bgl+) phenotype. One of the most common types of mutations giving rise to the Bgl+ phenotype is the transposition of IS elements like IS1 and IS5 into the bgl regulatory region bgl regulatory region or outside the bgl operon, which counteract the silencing of the operon The β-glucoside  of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which is responsible for the transport and phosphorylation of extracellular β-glucosides bglB, encodes a phospho-β-glucosidase which hydrolyzes the aglycon-sugar linkage in the phospho-β-glucosides that are transported and phosphorylated by BglF bgl operon.The bgl operon, such as the cyclic-AMP receptor protein (Crp), LeuO and BglJ bgl operon promoter by binding to a site in the regulatory region bgl regulatory region and increase the expression of the bgl operon bgl regulatory region and activates the operon bgl operon when bound to the bgl regulatory region, but these transcription factors have no effect on the region downstream of the promoter There are several transcriptional regulators reported to be involved in upregulating expression of the bgl operon is silent in wild-type E. coli cells. The major repressor of the bgl operon is the histone-like nucleoid structuring protein (H-NS), which is involved in chromosomal organization and acts as a global negative regulator As mentioned above, the bgl operon, H-NS binds close to the Crp-cAMP binding site within the regulatory region, as well as a downstream region within the bglG gene bgl operon are still not defined. In addition, it is still unclear what the exact mechanism is by which H-NS silences the bgl operon, although it is known that the binding of H-NS to both locations is required for full repression, and the repression involving H-NS at these two sites is synergistic bgl operon, StpA can function as a DNA-binding adapter of H-NS to repress the operon bgl operon when H-NS is absent bgl operon in wild-type E. coli cells.In relation to the gal operon, the binding of the galactose repressor  to the two operators in the gal operon alone is insufficient to repress the operon. Instead, the formation of a DNA loop, resulting from the protein–protein interaction between GalR proteins, is required for strong repression of the gal operon E. coli lac operon, with the binding of the lac repressor, LacI, to two of the three lac operators within the operon R and OL (separated by 2.3 kb) and loops them together by octamerization when the phage is incorporated into the E. coli chromosome DNA looping, mediated by transcription factors, is a mechanism sometimes used for strong and effective regulation of gene transcription hdeAB and rrnB operons in E. colibgl operon by H-NS, but this has not been demonstrated directly. Our prior studies have suggested that H-NS binding to its binding site(s) in the bgl promoter region is not sufficient to repress the operon, so we proposed that H-NS represses the bgl operon by the formation of a DNA loop between its two binding sites bgl operon repression was investigated in-depth in this study.As a common repressor, H-NS can exert its repressive effect on transcription by first binding to two or more DNA targets and then looping these target sites, trapping RNA polymerase at the promoter or excluding binding of RNA polymerase from its operator bglG gene are not required for bgl operon silencing as the operon remains uninducible when these terminator sequences are deleted. We then examine the effect of preventing the formation of the H-NS-mediated DNA loop on bgl operon transcriptional activity, as well as the ability of E. coli cells to grow on aromatic β-glucosides. In order to do so, the formation of the DNA loop was altered using the following approaches: (1) preventing H-NS self-oligomerization, (2) preventing or reducing H-NS binding in the bgl operon regulatory region, (3) preventing or reducing H-NS binding within the bglG gene, (4) changing the phase of the DNA between the two H-NS binding sites, and (5) changing the distance between the two H-NS binding sites. We demonstrate that H-NS binding to both upstream and downstream sites simultaneously is insufficient to abolish transcription. However, a DNA loop between these binding sites, formed by H-NS oligomerization, is conceivably required for full repression. The proposed H-NS-mediated DNA loop is robust as it is independent of DNA phasing and the length of intervening DNAs. The presence of such a strong DNA loop would explain why other transcription factors (reported to be involved in bgl regulation), when expressed at wild type levels, have negligible effects on bgl operon expression. These results provide a new perspective on the mechanism by which the bgl operon is repressed by H-NS in wild-type E. coli cells.Here, we first show both terminators flanking the 22.1bgl operon, bglG, is flanked by two Rho-independent terminator sequences. To determine if these terminators play a role in silencing the bgl operon, we first removed both terminators from our bgl operon reporter, Pbgl-bglG-lacZ strain The first gene of the bgl operon, yielding strain G50. As seen in − phenotype, as its colonies did not show a visible red color after 1 day of incubation on MacConkey indicator plates with 0.5% salicin at 37°C, indicating its inability to ferment salicin. Also, no visible colonies were seen on M9 minimal agar plates with salicin after 2 days of incubation at 37°C, which further confirmed its Bgl− phenotype. All of these results showed that the bgl operon remains silent  in the absence of both terminators flanking the bglG gene. This terminator-less strain was used in most of the experiments in this study.The two terminators were also removed from the native 2.2bgl operon by forming a DNA loop between its two binding sites bgl operon activity.In an earlier paper, we proposed that H-NS represses the hnsL30P mutant (a missense mutation in the hns gene leading to a change of leucine to proline at the 30th codon) (hnsL30P increased 550 times compared to strain BW25113 (0.08 units) which expresses the wild type H-NS, indicating this H-NSL30P mutant protein lost most of the repressive effect on the operon. However, the operon activity of hnsL30P was lower (by 53 units) than that of the Δhns mutant . Both Δhns and hnsL30P showed Bgl+ phenotypes on both MacConkey + salicin plates and M9 + salicin plates.We first determined the effect by preventing the oligomerization of H-NS using the h codon) A [29], . Thmutation . This cobglJ, leuO and rcsB in the background of G50, so that these positive regulators could  outcompete H-NS binding to the bgl regulatory region  deletion of the 3′ region, thought to contain the H-NS binding site; and (3) substitutions of the H-NS binding region with one or two TetR operators.Previous studies had shown that the downstream H-NS binding site is approximately within positions +468 to +607, relative to the bglG gene was removed from G50 (ΔbglG50) to ensure that no binding of H-NS to bglG could occur. As shown in bglG50 increased dramatically (200-fold) in comparison to G50, while ΔbglG50 shows a Bgl+ phenotype. These facts indicate that the H-NS binding site in the bglG gene is essential for bgl operon silencing by H-NS.The entire bglG50, deleted for the 3′ region, from +466 to the end of the gene, was used for the operon lacZ reporter expression , an over 5-fold increase of operon activity was observed compared to G50-Z which carries the complete bglG50 gene in front of the reporter  may be due to the less effective binding of H-NS to the remaining region of the truncated bglG gene.When a short version of reporter C. This fbglG gene with either one or two TetR operators, respectively  (Strains G50.1R and G50.2R were created by replacing part of the putative H-NS binding site within the ectively D. Both sxpressed E. These ctively) .2.5bgl operon, we first examined the impact of DNA phasing between the two H-NS binding sites on DNA loop formation . As shown in bglG50 gene. As expected, all of these modified strains with various sizes of DNA insertions within the bglG gene still showed Bgl− phenotypes using our standard assays involving MacConkey + salicin plates and M9 + salicin plates.To eliminate a possible effect of one or both of the two terminators on DNA loop formation, we used strain G50 deleted for both terminators. As shown in bglG50 gene, which may otherwise abolish transcription of downstream bglF and bglB, the hns mutation was individually transferred to G50, 1017-G50 and 1305-G50 . These new strains were examined for their BglB activities using our standard approach. As expected, a dramatically increased operon activity was seen for each strain, and there is little difference (10% or less) among these three new strains (bglG50. All of these results demonstrated that increasing distances between the two H-NS binding sites does not appreciably affect strong repression of the bgl operon by H-NS. This therefore indicates that H-NS-mediated DNA looping is robust.To further confirm the absence of a terminator sequence within those two DNA fragments (1017 bp and 1305 bp) inserted at the  strains , indicat3bglGFB operon using a DNA looping mechanism. As the major repressor, dimerized H-NS proteins have long been thought to bind to an upstream site within the regulatory region and a downstream site within the bglG gene. However, these bindings alone are not sufficient to abolish operon expression, although they do contribute to partial repression. Instead, by individually disrupting H-NS self-oligomerization or by preventing DNA binding to either of the two sites, we proposed that H-NS, when bound to both sites, bridges by loop formation the regulatory region and the bglG gene via oligomerization, forming the DNA loop and thus blocking transcription.In this work, we have provided evidence that the nucleoid structuring protein, H-NS, silences the N-terminal domain, leading to formation of higher-order oligomers bgl operon, H-NS is a strong dominant repressor which simultaneously binds to the bgl regulatory region and the 3′ region of the bglG gene bgl operon expression, as the repression is much greater when H-NS binds to both locations than when it binds to either one H-NS is characterized by self-dimerization and oligomerization via its bgl operon (bgl promoter and the 5’ region of bglG are embedded within the loop. Such an H-NS-mediated DNA loop efficiently abolishes the transcription of the bgl operon by (1) blocking the binding of RNA polymerase to the bgl promoter, and/or (2) trapping RNA polymerase when bound to the bgl promoter to prevent readthrough.In this work, we found that H-NS lost the vast majority (85%) of its repressive ability when it is deficient in self-oligomerization, even though it still maintains its DNA binding property. It is generally recognized that self-oligomerization is vital for H-NS to loop DNA l operon . Brieflybgl operon regulation, in addition to the downstream binding site within bglG, there may be some other unidentified intragenic or intergenic promoters (for sigma70 to bind) overlapped with H-NS binding sites in both sense and antisense orientations. Transcription (leading to non-coding RNAs) from these promoters could be (strongly) repressed by H-NS as well. Although the possible presence of such new promoters and their repression by H-NS contribute to our understanding of the complex regulation of the bgl operon, these facts should not replace the main DNA looping mechanism by which H-NS silences the operon.Instead of usually repressing canonical transcription, Singh et al. reported that H-NS primarily functions to repress spurious transcription of non-coding RNAs within the genes previously described as being H-NS repressed bgl operon, the proU operon, encoding an osmoprotectant uptake system, is subject to strong H-NS repression as well proV of the operon proU repression by H-NS bgl system would most likely be applicable to the proU operon.Similar to the bgl operon as to its silencing state and its non-inducibility in wild-type cells. It is also conceivable that with such a stable DNA loop, involving the entire promoter region, other transcription factors, such as BglJ, RcsB, LeuO and Crp, when expressed at the wild-type levels, either fail to access or are trapped in the promoter region, thus being essentially without effects on bgl operon expression. When these proteins are produced in large amounts or have better binding sites, the binding of H-NS to the upstream site may be partially prevented, thereby elevating rates of transcription.Alterations of DNA phasing and the distances between two binding sites have negligible effects on H-NS-mediated looping, indicating that the formed DNA loop is exceptionally stable. This may be correlated to the large size (about 1 kb nucleotides) of the DNA loop, which confers rotational flexibility. The presence of such a robust DNA loop would definitely contribute to our understanding of the + (able to utilize β-glucoside for growth) + in previous studies, were not able to grow on minimal agar plates with salicin as the sole carbon source. In this report, we show that overproduction of BglJ, RcsB, the BglJ/RcsB complex or LeuO did not give rise to appreciable growth on minimal salicin agar media although visible red color could be seen at the centers of some of their colonies on MacConkey agar plates containing salicin. Therefore, phenotypic results obtained from MacConkey indicator plates with salicin were sometimes misleading. As a result, strains considered to have a Bgl+ phenotype in this study must fulfill two requirements: (1) appearance of colonies with visible red color on MacConkey agar plates containing 0.5% salicin after 1 day of incubation at 37°C, and (2) appearance of colonies of visible sizes on M9 agar plates with salicin as the sole source of carbon after 2 days of incubation at 37°C.Indicator agar plates such as those containing MacConkey agar with salicin or bromothymol blue with salicin have been used to determine the phenotypes of growth/fermentation on/of β-glucosides, with a dark color (red or blue) developed within individual colonies that are BglbglG gene are two terminators that effectively block transcription due to the formation of hairpin-like secondary structures in the absence of the activated antiterminator, BglG bgl operon silencing. Using a mutant strain (G50) only deleted for these two terminators with all other regions unchanged, we showed that its operon activity increased only slightly compared to the wild-type, and it showed a Bgl− phenotype (bglG do not exert an appreciable effect on the silencing of the bgl operon in wild-type E. coli cells. We thus distinguished the roles of H-NS and both terminators with respect to their regulatory effects on bgl operon expression. Strain G50 (without the two bgl terminators) was used throughout this study, showing that H-NS is still able to form a DNA loop, thus silencing bgl operon expression, when the terminator structures are not present.Flanking the henotype B. This ibgl operon transcriptional activities under H-NS repression . As RcsB is a global regulator, overexpression of rcsB might have caused a pleiotropic effect on transcription of other genes and cell growth in addition to affecting bgl operon transcription Overexpression of positive regulators, BglJ and LeuO, increased pression F. This ipression F. As Bglbgl operon expression was seen when stronger Crp-cAMP binding to the bgl regulatory region occurred. However, all strains, either producing higher levels of positive regulators or carrying a better binding site, are still Bgl− due to the low levels of operon expression  the binding of TetR to two operators within the bglG gene further impeded H-NS binding to its target site, reducing the H-NS looping ability and increasing transcription, even though the bound TetR might act as a roadblock, blocking transcription; and (3) the region replaced by the TetR binding sites might not be the actual H-NS binding site within the bglG gene, or H-NS might be able to bind to (an)other location(s) in the gene as deletions of the entire bglG gene or a larger portion of the gene either eliminated or substantially disrupted DNA looping.Previous studies had reported that H-NS binds to a region approximately 600 to 700 base pairs downstream of the  control E. Howeve+ mutations mediated by small IS transposons are positively regulated by the level of the anti-terminator protein, BglG bgl operon using the 3C technology bgl operon in the various types of live E. coli cells used in this study Our recent work revealed that the frequency of Bgl44.1E. coli K12 strain BW25113 tet promoter, Ptet4.2bgl promoter (Pbgl) and the bglG gene plus the bglG-flanking terminators was replaced by a cat gene amplified from pKD3 cat gene was flipped out by pCP20, yielding strain ΔPbglG, in which both Pbgl and bglG with the two terminators were replaced by an 85-bp FRT scar. This strain was then used as a beginning strain to make several other strains.Using wild type strain BW25113, the chromosomal region containing the 4.3bgl operon, bglG, is flanked by two transcriptional terminators. To determine if these terminators are important for H-NS mediated bgl operon silencing, they were deleted from the chromosome. To do this, the strain ΔPbglG was used as the starting strain. Present in plasmid pKES50 bgl-bglG50-lacZ cassette consisted of the lacZ gene  fused to a region downstream of the bgl promoter (Pbgl), a modified bglG gene, bglG50, . The Pbgl-bglG50 DNA region together with the downstream lacZ’s RBS (between bglG50 and lacZ) was amplified from plasmid pKES50 using oligos Pbgl-Xh-Fbg and bglG-Bm-Rm . This yielded pKDT_Pbgl-G50, in which the native bgl promoter drives the terminator-less bglG gene (bglG50). The cassette containing the kmr gene, rrnBT and Pbgl-G50 (kmr:rrnBT:Pbgl-G50) was amplified from pKDT_Pbgl-G50 using oligos Pbgl.G50-P1 and bglF.G50-P2  followed by bglF and bglB. The intergenic region (aatagcttcacaggaaacagct) between bglG and bglF is the same as one between bglG and lacZ in pKES50 lacZ’s RBS for bglF.The first gene in the lG-Bm-Rm . The PCRF.G50-P2 . The PCRtetR gene and a spectinomycin resistance (spr) marker was transferred to G50 by P1 transduction as mentioned above, yielding strain RIG50. Also, the deletion of hns was transferred to G50 by P1 transduction, yielding ΔhnsG50.The strain G50 was used as the recipient strain for P1 transduction in order to make other strains. First, the transcription unit including a constitutively expressed 4.4bgl operon, one within the bgl regulatory region and the other within the bglG gene, although exact binding sites have not been defined bgl regulatory region, the upstream region  carrying the possible H-NS binding site in G50, was first replaced by a kmr gene amplified from pKD4 using oligos Pbglcat-P1 and Pbgl2-P2  in Pbgl resulted from a C to T substitution at position −134 relative to the bglG translational initiation site bgl_O*Crp, driving bglG expression, was integrated upstream of bglF in ΔPbglG cells, yielding strain G51. This strain is the same as G50 except that it carries a stronger Crp-cAMP binding site in the bgl promoter region.To make strain G51, plasmid pKES51 4.7tetkmr:rrnBT:Ptet”, containing the kmr gene, the rrnB terminator (rrnBT) and the Ptet promoter, was amplified using the primer pair Ptet-bglJ-P1 and Ptet-bglJ-P2 (bgl terminators) to replace the yjjQ promoter . Chromosomal integration was confirmed, first by colony PCR, and subsequently by DNA sequencing. This yielded strain G50Ptet-bglJ, in which the strong tet promoter drives the yjjQ-bglJ operon. Similarly, the “kmr:rrnBT:Ptet” cassette amplified by Ptet-leuO-P1 and Ptet-leuO-P2 , yielding strain G50Ptet-leuO. The same cassette amplified by Ptet-rcsB-P1 and Ptet-rcsB-P2 , yielding strain G50Ptet-rcsB.Using plasmid pKDT:P-bglJ-P2 . The PCR-leuO-P2  from pKD-rcsB-P2  was subsbglJ/rcsB double overexpression strain, the kmr gene was first flipped out from G50Ptet-bglJ by pCP20, yielding G50Ptet-bglJ-kms, which is sensitive to kanamycin. The rcsB gene, driven by the strong tet promoter with a kmr marker, was then transferred to G50Ptet-bglJ-kms by P1 transduction, yielding strain G50Ptet-bglJ/rcsB. The rcsB mutation was transferred to G50Ptet-bglJ-kms as well, yielding strain G50Ptet-bglJΔrcsB, in which the strong tet promoter drives bglJ in the absence of rcsB.To make a 4.8bglG gene, the entire bglG gene and its two flanked terminators were deleted. First, the region from −78 to +946 relative to the bglG translational initiation site, containing the first terminator, the bglG gene and the second terminator, was replaced by a kmr gene amplified from pKD4 kmr gene was then flipped out using pCP20, yielding strain ΔbglG50, in which the bglG gene with the two flanking terminators was replaced by an 85 bp fragment.To completely abolish H-NS binding in the 4.9bglG gene, which is located at +479 to +606 relative to the bglG translational initiation site bglG translational initiation site, was replaced by one TetR operator with sequence “tccctatcagtgatagaga” tetR-spr cassette (constitutively expressing tetR) was transferred from BW-RI to G50.1R and G50.2R, yielding strains RIG50.1R and RIG50.2R, respectively.In vitro protein/DNA binding assays showed that H-NS proteins are capable of binding to a region within the 4.10bgl operon, formed via H-NS protein oligomerization, is DNA phase-dependent, a 5-bp fragment with nucleotides “ATAGA” and a 10-bp fragment with nucleotides “ATAGAATAGA” were inserted individually into the bglG gene between +108 and +109 relative to the bglG translational initiation site in strain G50. Briefly, these two small fragments (5 bp and 10 bp) were first integrated into the designated locus (between +108 and +109) within the bglG50 gene in plasmid pKDT_Pbgl-G50 using fusion PCR, yielding plasmids pKDT_Pbgl-G50.5 bp and pKDT_Pbgl-G50.10 bp, respectively. Using these plasmids as templates, the “kmr:rrnBT:Pbgl-G50.P5” cassette and the “kmr:rrnBT:Pbgl-G50.P10” cassette were amplified using oligos Pbgl.G50-P1 and bglF.G50-P2 , encoding kanamycin resistance, was amplified from pKD13 and subsequently inserted into the same location as for 1017-G50, yielding 1305-G50 that carries a 1305 bp fragment between the two H-NS binding sites.To determine if the stability of H-NS-mediated looping in the hns by P1 transduction, yielding strains Δhns-1017-G50 and Δhns-1305-G50.The two strains, 1017-G50 and 1305-G50, were used as the recipient strains for transferring the deletion of 4.12bgl operon LacZ reporter, which is composed of the bglGFB promoter, the first terminator, bglG, the bglG/bglF intergenic region (with the second terminator) and the first 9 codons of bglF, plus a stop codon at the 3′ end, immediately upstream of the lacZ’s RBS at the lac locus. A kmr gene followed by a rrnB terminator is located upstream of this reporter. Using the same protocol for strain BW25113_Z construction kmr:rrnBT:Pbgl-bglG50” on plasmid pKDT_Pbgl-G50 and “kmr:rrnBT:Pbgl-bglG51” on plasmid pKDT_Pbgl-G51 were individually integrated into the chromosome of the default strain MGIBKY lacI gene and the lacI/lacZ intergenic region, yielding strains G50-Z and G51-Z. These two reporters were transferred into a Δcrp Glp+ mutant (able to grow on glycerol) crpG50-Z and ΔcrpG51-Z, respectively.Strain BW25113_Z bgl-bglG50-lacZ operon reporter was transferred from G50-Z to strains G50Ptet-bglJ, G50Ptet-leuO, G50Ptet-rcsB, G50Ptet-bglJ/rcsB and G50Ptet-bglJΔrcsB, yielding G50-Z-Ptet-bglJ, G50-Z-Ptet-leuO, G50-Z-Ptet-rcsB G50-Z-Ptet-bglJ/rcsB and G50-Z-Ptet-bglJΔrcsB, respectively.To determine the effects of overexpression of positive regulators on H-NS binding to the upstream site within the regulatory region, the PbglG gene, which is located between +479 and +606 relative to the translational initiation site of bglG. To examine the effect of this region on β-galactosidase (LacZ) activity, part of this region of the bglG50 gene in plasmid pKDT_Pbgl-G50 was replaced by one or two operators of TetR using fusion PCR, yielding pKDT_Pbgl-G50.1R and pKDT_Pbgl-G50.2R, respectively. The cassette of “kmr:rrnBT:Pbgl-bglG50.1R” on pKDT_Pbgl-G50.1R and the cassette of “kmr:rrnBT:Pbgl-bglG50.2R” on pKDT_Pbgl-G50.2R were individually transferred to the same location on the chromosome as for G50-Z, yielding strains G50.1R-Z and G50.2R-Z, respectively. The two operon reporters are the same as G50-Z, except that part of the H-NS binding site within bglG was replaced by one or two TetR operators. Using G50.1R-Z and G50.2R-Z as recipient strains, two new operon reporters were made by transferring the tetR expression unit with a spr marker using P1 transduction, yielding RIG50.1R-Z and RIG50.2R-Z, respectively.As described above, H-NS proteins are capable of binding to a region within the bglG50, a new operon reporter similar to G50-Z, carrying the bgl promoter and only the 5′ region of bglG50 with no 3′ region, was made. The cassette of “kmr:rrnBT:Pbgl-bglG50’” , amplified from pKDT_Pbgl-G50 by oligos P1n-P1n and G50tn-Z-P2  added to the end of bglG50’, and the RBS for lacZ remains the same as for G50-Z.To further prevent H-NS binding to 0tn-Z-P2 , was fusbglG when bound to it, another new lacZ transcriptional reporter was made in which the bglG-lacZ fusion is driven by a constitutive promoter. The cassette, “kmr:rrnBT:Iq-bglG” (with lacIq driving bglG expression), was amplified from strain Iq-G lacZ as for other operon lacZ reporters at the lac locus. This reporter, carrying a lacIq constitutive promoter driving the terminator-less bglG gene that is transcriptionally fused to a lacZ gene, was transferred into strains Δhns and hnsL30p, yielding Δhns_Iq-G-Z and hnsL30P_Iq-G-Z, respectively. Similarly, the cassette “kmr:rrnBT:Ptet” from strain Ptet-G lacZ and subsequently transferred to Δhns and hnsL30P, yielding Δhns_Ptet-G-Z and hnsL30P_Ptet-G-Z, respectively.To determine if H-NS or H-NSL30P has an inhibitory effect on expression of 4.13E. coli cells were grown on MacConkey indicator plates with 0.5% salicin at 37°C overnight. Visible red color was shown for colonies with a Bgl+ phenotype, indicating their ability to ferment salicin. Otherwise, colonies with a Bgl− phenotype remained white in color. In addition, cells were grown on M9 minimal agar plates with 0.5% salicin as the sole carbon source at 37°C for 2 days to further confirm their phenotype. To have a Bgl+ phenotype, cells should form visible colonies during the 2-day incubation. Otherwise, the cells are considered to be Bgl−. In this study, we define a Bgl+ phenotype as the appearance of red colonies on MacConkey + salicin plates during a 24-hr incubation plus the formation of visible colonies on M9 + salicin plates during a 48-hr incubation.4.14600 of 0.025. The cells were grown at 37°C with shaking. Five samples with 0.8 mL each were collected during the late exponential growth phase when the bgl operon was fully induced with an OD600 of above 1.5. The samples were centrifuged at a speed of 5,500 rpm for 2.5 mins. The supernatant was removed, and the cells were suspended with 1 mL of Z-buffer with 50 µg/mL chloramphenicol.The protocols for β-glucosidase assay as described in previous studies were followed with minor changes p-nitrophenyl-β-d-glucoside  were added to the cell suspension in Z-buffer. After a visible yellow color appeared, 0.5 mL of 1 M sodium carbonate was added and vortexed to stop the reaction. The reaction mixture was diluted, if appropriate, and centrifuged at a speed of 15,000 rpm for 2.5 mins. The absorbance of the reaction mixture was measured at 420 nm and 550 nm. The BglB activity of each sample was calculated using the equation: β-glucosidase (BglB) activity = [1000 × (OD420nm – 1.75 × OD550nm) × Dilution factor]/[OD600nm × Time of reaction (min) × Volume of sample (mL)]. The activity of the strain was determined by averaging the BglB activities of the samples measured.The samples were warmed in a 37°C water bath. To start the reaction, 200 µL of 4.15600 of 0.03. The cells were grown at 37°C with shaking. Four samples were collected during the exponential growth phase, when the OD600 reaches around 0.2, 0.4, 0.6 and 0.8. Collected samples could be kept frozen at −20°C prior to the measurements.Reporter strains were grown in LB medium at 37°C with shaking. After at least 6 hrs, 20 µL of the culture were transferred to 3 mL of M63 minimal media with 0.5% glycerol. Then it was left overnight for growth at 37°C with shaking. The overnight culture was inoculated into 5 mL of M63 minimal medium with 0.5% glycerol, with a starting ODo-nitrophenyl-β-d-galactopyranoside (β-ONPG) was added to each sample. After a visible yellow color appeared in some tubes, 0.5 mL of 1 M sodium carbonate was added and vortexed to stop the reaction. The reaction mixtures were diluted, if appropriate, and centrifuged at a speed of 15,000 rpm for 2.5 mins. The absorbance of the reaction mixture was measured at 420 nm and 550 nm. The LacZ activity of each sample was calculated using the equation 420nm – 1.75 × OD550nm) × Dilution factor]/[Time of reaction (min) × Volume of sample (mL)]. The activity of the strain was determined by the slope of the LacZ activities versus cell densities (OD600) of the four samples collected at different times.200 µL of samples and 800 µL of Z-buffer were mixed with 25 µL of chloroform by vortexing for 10 s twice. The samples were warmed in a 37°C water bath. To start the reaction, 200 µL of Katie Jing Kay Lam: Methodology, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Zhongge Zhang: Conceptualization, Methodology, Data curation, Writing – original draft, Writing – review & editing, Supervision. Milton H. Saier: Conceptualization, Writing – review & editing, Supervision, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Mrnip−/− spermatocytes have impaired DNA double‐strand break (DSB) repair, they display reduced sex body formation and defective MSCI. We show that Mrnip−/− undergoes critical meiocyte loss at the diplotene stage. Furthermore, we determine that DNA DSBs (induced by SPO11) and synapsis initiation (facilitated by SYCP1) precede Mrnip expression in testes. Altogether, our findings indicate that in addition to an emerging role in DNA DSB repair, MRNIP has an essential function in spermatogenesis during meiosis I by forming drop‐like accumulations interacting with the sex body.Meiosis has a principal role in sexual reproduction to generate haploid gametes in both sexes. During meiosis, the cell nucleus hosts a dynamic environment where some genes are transcriptionally activated, and some are inactivated at the same time. This becomes possible through subnuclear compartmentalization. The sex body, sequestering X and Y chromosomes during male meiosis and creating an environment for the meiotic sex chromosome inactivation (MSCI) is one of the best known and studied subnuclear compartments. Herein, we show that MRNIP forms droplet‐like accumulations that fuse together to create a distinct subnuclear compartment that partially overlaps with the sex body chromatin during diplotene. We demonstrate that  ATMataxia telangiectasia mutatedATRataxia telangiectasia and Rad3 relatedBrca1breast cancer 1, early onsetCONcontrolDdx3xDEAD‐Box Helicase 3, X‐linkedDdx3yDEAD‐Box Helicase 3, Y‐linkedDMC1DNA meiotic recombinase 1DSBDNA double‐strand breakFthl17ferritin heavy polypeptide‐like 17H1Ttesticular H1 histoneH3S10pPhospho‐Histone H3 phosphorylation at serine 10HETheterozygoteHormad1HORMA domain‐containing 1Hormad2HORMA domain‐containing 2KAP1KRAB‐associated protein‐1KOknockoutKOMPknockout mouse projectMdc1mediator of DNA damage checkpoint 1MLH1MutL homolog 1MLH3MutL homolog 3MRN complexMre11 Rad50 Nbs1MRNIPMre11 Rad50 Nbs1 interacting proteinMSCImeiotic sex chromosome inactivationNBS1NibrinPpostnatal dayParp2poly (ADP‐ribose) polymerase family member2PCNAproliferating cell nuclear antigenPgk1phosphoglycerate kinase 1Pgk2phosphoglycerate kinase 2RAD50DNA repair protein Rad50RAD51DNA repair protein Rad51 homolog 1RbmyRNA‐binding motif protein, Y chromosomeRNA POL IIRNA Polymerase IIRnf212ring finger protein 212ssDNAsingle stranded DNASYCP1synaptonemal complex protein 1SYCP3synaptonemal complex protein 3Tex11Testis expressed gene 11Ube1y1ubiquitin‐activating enzyme, Y chromosomeUsp26ubiquitin specific peptidase 26Utp14aUTP14A small subunit processome componentUtp14bUTP14B small subunit processome componentγH2AXH2A histone family member X phosphorylated at serine 1391Meiosis is an essential process of sexual reproduction, involving one round of DNA replication and two rounds of chromosome segregation leading to the production of haploid gametes during meiosis I and II.Meiotic recombination, the reciprocal exchange of chromosomal DNA between parental chromosomes, is initiated in the leptotene stage of prophase I by the formation of programmed DSBs by topoisomerase II‐like enzyme SPO11.Alignment of the homologous chromosomes begins at the leptotene stage when two pairs of sister chromatids form axial elements through association with a proteinaceous structure called the synaptonemal complex. First, Synaptonemal complex protein 3 (SYCP3) starts to localize on the lateral axial elements of the synaptonemal complex. Later, during the zygotene stage, Synaptonemal complex protein 1 (SYCP1) begins to localize in transverse elements of homologous chromosomes. Formation of the synaptonemal complex is required for homologous recombination to occur in the pachytene stage.Brca1, H2ax, Atr, Parp2, Mdc1, and Hormad2 leads to mis‐expression of lethal sex‐linked genes followed by severe defects in meiotic progression or arrest.The occurrence of DSBs leads to one more important event, the phosphorylation of the histone variant H2AX at serine 139 (γH2AX) during the leptotene stage of prophase I.Mrnip knockout mouse model has shown that male mice were subfertile and states that Mrnip is probably responsible for homologous recombination through its interaction with the MRN complex in meiosis.Mrnip contributes to the progression through meiosis, and what upstream genetic factors regulate Mrnip expression.The MRN complex interacting protein (MRNIP) was discovered as a protein involved in DNA DSB repair in somatic cells through interaction with MRN complex proteins.Mrnip deletion leads to compromised DNA DSB repair, reduced sex body formation, and defective MSCI. Additionally, we identified that Spo11 and Sycp1 are critical for downstream Mrnip expression. Overall, Mrnip is essential for meiotic progression, spermatogenesis, and male fertility in mice.In this study, we report that MRNIP is expressed in mid‐pachytene to diplotene spermatocytes and becomes partially associated with sex body chromatin. We show that 22.1tm1a(EUCOMM)Wtsi (herein referred to as Mrnip+/−) mice were acquired from the Wellcome Trust Sanger Institute's knockout mouse project and maintained in a mixed C57BL/6J, (C57BL/6N)/129SvEv background. The null allele in these mice was generated through splicing to a LacZ trapping element present in the targeting cassette.MrnipMrnip mice were housed in a specific pathogen‐free animal facility in individually ventilated cages under controlled conditions of light (12 h light/12 h dark). Animal handling was conducted in accordance with: the Institutional Animal Care and Use Committees of Baylor College of Medicine, Houston, USA; the Research Institute of Microbial Disease at Osaka University; the Finnish Animal Ethics Committee license (34/2018) and the institutional animal care policies, which fully meet the requirements of European Union Directive 2010/63/EU and the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes . Genotyping primers are available in Table Spo11 and Sycp1 mice were housed in the Animal Care Facility at the University of Illinois at Urbana‐Champaign, Urbana, IL, USA under 12 h dark/12 h light cycles and at 22 ± 1°C. Animal handling and procedures were approved by the UIUC Institutional Animal Care and Use Committee. The genotyping was done as published earlier.2.2Testes were fixed in Bouin's Fixative and stained with periodic acid‐Schiff's reagent  as described in Ref. . The secAdult male testes were fixed in 2% paraformaldehyde (PFA)/PBS for 3 h at 4°C, then washed briefly in PBS, placed in 30% Sucrose/PBS, followed by incubation in a 1:1 mixture of 30% Sucrose/PBS: OCT. Testes were embedded in OCT, frozen on dry ice, and sectioned (10 μm). Control and KO sections were placed on the same slide and stored at −80°C. The primary and secondary antibodies and dilutions are available in Table Immunofluorescent staining with paraffin‐embedded specimens was also done as described previously2.3TUNEL assay for apoptotic cell detection was performed according to the manufacturer's instructions (Roche). Briefly, testes were fixed in 4% PFA, dehydrated through an ethanol series, embedded in paraffin, sectioned at 5 μm, and stained according to the assay instruction. Testes from three mice of each genotype were analyzed. Apoptotic cells were calculated in at least 100 seminiferous tubule cross‐sections and three sections per testis. The result is presented as an average of apoptotic cells per 100 tubules.2.4g). The supernatant was removed, and pellet resuspended in 1 ml of hypotonic buffer  supplemented with dithiothreitol (0.5 μM) and incubated for 30 min at RT protected from light. The cell suspension was centrifuged for 7 min (100× g), the supernatant was removed, and the cell pellet was gently resuspended in 0.3–1 ml of 100 mM sucrose solution . The objective slides (Super Frost Plus (Fisher Scientific)) were dipped in 1% PFA  and 12 μl of cell suspension was applied to the PFA‐coated slide dropwise and then allowed to spread by tilting the slide. The slides were kept in a humidified chamber for 2 h, then air‐dried. Prepared nuclear spreads were stored at −80°C.Testicular nuclear spreads were prepared as described in Ref.  with somBefore proceeding to immunostaining, the spreads were air‐dried for 20 min at RT and washed with 1XPBST . Blocking was done with 5% fetal bovine and 5% goat serum for 1 h, followed by overnight incubation with primary antibodies, three PBST washes, and 1 h incubation with secondary antibody was performed. The primary and secondary antibodies are listed in Table 2.5g for 5 min at room temperature. The supernatant was discarded and 3 ml of 1% sodium citrate was added and centrifuged as described above.Adult mouse testes were harvested from control and knockout mice respectively and treated using a modified protocol published by Evans et al.Mrnip−/− mice, respectively.The cells were incubated overnight at +4°C in 3:1 methanol: glacial acetic acid fixative. Fresh fixative was added to the cells before slide preparation. The slides were prepared using a modified previously published protocol.2.6Gapdh was used for normalization by the CT method.Mouse cDNA was prepared from multiple adult tissues of C57BL6J/129SvEv hybrid mice and testes. Briefly, tissues were dissected and snap frozen in liquid nitrogen. RNA was extracted using TRIzol (Invitrogen) or RNeasy Protect Mini kit (Qiagen) following the manufacturer's protocol. 0.8–1 μg RNA was transcribed to cDNA using First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) or qSCRIPT cDNA supermix (Quanta) following the manufacturer's protocol. The generated cDNA was used to perform reverse transcription‐ (RT‐PCR) and quantitative real‐ time‐polymerase chain reaction (qRT‐PCR). The primers used are listed in Table 2.7) and Mrnip−/− male mice were housed with wild‐type females for at least 3 months. Copulation was confirmed by checking for vaginal plugs and the number of pups in each cage was recorded.Sexually mature control  media supplemented with bovine serum albumin (0.3 mg/ml) at 37°C (capacitating conditions). Sperm samples were diluted, analyzed, and imaged using Hamilton Thorne's CEROSII sperm analysis system .Sperm motility was analyzed as described earlier.2.9Testes were dissected and snap frozen in liquid nitrogen. Tissues were solubilized in T‐per (Thermo Fisher) lysis buffer supplemented with protease and phosphatase inhibitors  and PhosSTOP (Roche) or in 1XTBS with 1% Triton X‐100 and protease inhibitors (Pierce). We used Bis‐tris 4%–12% pre‐cast gels (Invitrogen) or 12.5% hand‐casted polyacrylamide gels to load 20 μg of total protein. Proteins were transferred to nitrocellulose membranes. Blocking was done in 5% BSA for phospho antibodies Table  and in 52.10t‐test  by Microsoft Excel and GraphPad Prism 9.3.1 . Data represents the mean ± standard deviation (±SD). At least three mice of each genotype were used in each experiment unless indicated differently.Statistical analysis was performed using a two‐tailed Student's 33.1Mrnip gene has seven exons and is located on chromosome 11. Our RT‐PCR analysis demonstrates that Mrnip is ubiquitously expressed in multiple mouse tissues with the strongest expression in the kidney, brain, and testis  day 5 testes and onwards as shown by RT‐PCR of mouse testes at different time points Wtsi (herein referred to as Mrnip+/− for heterozygous and Mrnip−/− for homozygous) from the Knockout Mouse Project  pups per litter, whereas Mrnip−/− males did not sire any pups  control and knockout e Figure . Notablye Figure . Mature e Movies  and S2. l Figure . Occasios Figure , insert.e Figure  and S2.3.5Mrnip gene expression was detected in the ovaries and uterus  and 6.7 ± 2.3 , respectively  12.5, E13.5, E16.5, newborn, and adult ovaries and testes  nuclear spreads in comparison to control  and Mrnip−/− , we have analyzed if ATM expression was influenced. The analysis by Western blot showed no clear changes in ATM expression in response to Mrnip deletion in testis lysate , which controls DNA repair in heterochromatin,− Figure . KAP1 ale Figure .n = 181) and Mrnip−/−  autosomes were aligned, whereas sex chromosomes were still unsynapsed as expected both in control and Mrnip−/−  of Mrnip−/− as compared to 3.58% ±2.71 of control (n = 286) spermatocytes  and Mrnip−/− 90.15% ±4.93 (n = 329) when the sex body was formed.Immunostaining of testis cross‐sections stained with γH2AX, a marker of the DNA damage response at DNA double‐strand breaks in early stages of meiosis, and later specific to sex bodiess Figure . Aberrans Figure . QuantifMrnip's role in MSCI, we performed qRT‐PCR of genes located specifically on X and Y chromosomes using adult testis cDNA. We compared the expression of Y‐linked  and X‐linked  genes that were shown to be associated with MSCI, and autosomal  genes as controls.Mlh1 gene (Chr 9) expressed in germ cells served as an indicator that cells had an overall non‐reduced transcription. Our data showed that most of the analyzed X‐ and Y‐linked genes expression remained active in Mrnip−/− mice  as compared to the control  exclusion is observed in non‐transcribing sex bodies and inappropriate exclusion is often associated with defective MSCI.I Figure . Quantif3.9Mrnip−/− testes, however very few, as evidenced by H1T antibody staining  localizes to the cross‐over sites in meiotic chromosomes.Mrnip−/− cells  in comparison to control  as compared to the control   labels chromatin of meiotic spermatocytes in prophase I diplotene and metaphase where it is associated with chromosome condensation and is not detected afterward in spermatids.r Figure . Further) Figure  as expecMrnip−/− chromosomes form bivalents, we quantified the presence of univalent and bivalent chromosomes in metaphase I spreads in control and Mrnip−/− testes , a transverse axial element protein of the synaptonemal complex,s Figure .Rnf212), a SUMO ligase required for crossing‐over formation.Rnf212−/− testes and that MRNIP colocalizes with H1T in mid‐pachytene to diplotene stage spermatocytes  during DNA DSB repair after gamma radiation in human cell lines and named the protein accordingly.Mrnip expression starts later during meiosis, the expression levels of multiple DNA DSB repair proteins  were decreased in Mrnip−/− testes lysates. Along with this, RAD51 foci counts were moderately increased in pachytene spermatocyte autosomes indicating the presence of unrepaired DNA DSBs. Given that the major DNA DSB repair events are taking place prior to MRNIP expression in spermatocytes, it is likely that during meiosis MRNIP exerts its effect on DNA DSB repair mostly through an indirect, yet uncharacterized feedback mechanism.Herein, we demonstrated that the deletion of the Mrnip−/−mice. This could suggest reduced formation of chiasmata. If this were the case, then chromosome bivalent formation at meiotic metaphase would likely be disturbed, which was not the case in remaining Mrnip−/− meiocytes that have reached metaphase. However, even though the cells bypassed earlier meiotic checkpoints and initiated metaphase, they often fail to align on the spindle and very few cells continued differentiation in Mrnip−/−.MLH1 protein localizes to meiotic cross‐over sites and marks the chiasmata positions.Mrnip−/− testes. This implies MRNIP is not necessarily playing a crucial role as a replication fork protection factor during meiosis as it does in the case of gamma radiation induced DNA DSB repair in somatic cells.We showed that PCNA and MRNIP expression during meiosis, overlapped only in mid‐pachytene stage spermatocytes, when MRNIP expression begins and PCNA expression decreases. The PCNA expression levels and patterns were not changed in Mrnip−/−. This alone could hardly account for such drastic spermatocyte loss, whereas an association with the lethal effects of non‐repressed genes from X and Y chromosomes could explain the massive loss of post‐pachytene spermatocytes in Mrnip−/− testes. Indeed, our study revealed that many genes on X and Y chromosomes were still transcriptionally active in Mrnip−/− instead of being silenced as in controls. Additionally, we showed that retrogene repression and de‐repression were altered in Mrnip−/− testes. Pgk1 and Utp14a expressed on the X chromosome remained active in Mrnip−/− testes, whereas they were already inactivated in the control. Consequently, retrogenes Pgk2 and Utp14b expressed on autosomes remained silenced in Mrnip−/− testes, instead of becoming de‐repressed as in the control to balance the missing function of the repressed gene. This repression and de‐repression sequence is altered in Mrnip−/− meiocytes. The loss of function of at least one such retrogenes has been shown to lead to spermatogenic arrest.Failure in sex body formation and RNA Pol II exclusion from sex body are morphological features often associated with defects in X and Y chromatin silencing.Brca1, H2ax, Atr, Parp2, Mdc1, Hormad1, Hormad2, and Sycp3.Accumulating studies propose that meiotic silencing involves many genes associated with the mitotic DNA damage response such as Mrnip is associated with DNA DSB repair in somatic cellsMrnip's association with a role in MSCI. However, Mrnip does not follow the classical pattern seen in other genes associated with MSCI, where defective synapsis and stage IV pachytene meiocyte loss is seen.Mrnip−/− synapsis was normal and this, likely, allowed meiocytes to survive through stage IV, mid‐ and late‐pachytene. This seemingly normal synapsis could be explained by the timing of MRNIP expression in meiocytes, starting at mid‐pachytene when the synapsing process and SYCP1 expression are coming to an end. The major loss of meiocytes in Mrnip−/− took place during the diplotene stage, likely due to insufficient MSCI.Earlier studies showed that Mrnip knockout chromosomes synapsed normally but MSCI was faulty, we are tempted to hypothesize that they could be two separate checkpoints; however, this requires further verification. It is important to note that a recent study by Lin et al. showed a reduced count of pachytene meiocytes and unsynapsed chromosomes in Mrnip−/−. However, it was difficult to judge the severity of the defect in the presented mouse model since quantification of synapsed versus non‐synapsed chromosomes was not included.There is an ongoing discussion whether the synapsis checkpoint and the MSCI checkpoint are two separate checkpoints or if MSCI is a secondary event followed by failure in synapsis.Mrnip expression during male meiosis. First, Spo11, of which expression is essential for double‐strand break formation and subsequent DNA recombination and homologous pairing during meiosis,Mrnip expression initiation in mouse testes. This suggests that DNA DSBs are one of the triggers necessary for Mrnip expression. Another critical event during meiosis is synaptonemal complex formation; specifically, Sycp1 expression which promotes homologous recombination during meiosis.Sycp1 expression is vital for the initiation of Mrnip expression.Furthermore, our study identified two critical upstream genes preceding Mrnip function during meiosis, Figure Mrnip function causes infertility in male mice. Finally, the presented data raise the possibility that impaired function of MRNIP could be a cause of infertility in humans and other species.In conclusion, our study shows that MRNIP is a specific spermatocyte biomarker of mid‐pachytene through diplotene stages. We demonstrate that MRNIP staining partially overlaps with the sex body in diplotene. Furthermore, we showed that MRNIP is required for proper DNA DSB repair protein function, efficient meiotic sex chromosome inactivation, and full‐fledged spermatogenesis (schematic representation of key features of Martin M. Matzuk and Ramiro Ramirez‐Solis initiated the study; Renata Prunskaite‐Hyyryläinen and Julio M. Castañeda designed research; Samina Kazi, Renata Prunskaite‐Hyyryläinen, Julio M. Castañeda, Audrey Savolainen, Yiding Xu, Ning Liu, Huanyu Qiao, Kaori Nozawa, and Zhifeng Yu performed research; Samina Kazi, and Renata Prunskaite‐Hyyryläinen analyzed data; Renata Prunskaite‐Hyyryläinen and Samina Kazi, wrote the article; Renata Prunskaite‐Hyyryläinen and Martin M. Matzuk acquired grants to support the study.The authors have no conflicts of interest to declare.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Video S1Click here for additional data file.Video S2Click here for additional data file.Text S1Click here for additional data file.


Escherichia coli K-12 strains represent perhaps the best known model bacteria, we do not know the identity or functions of all of their transcription factors (TFs). It is now possible to systematically discover the physiological function of TFs in E. coli BW25113 using a set of synergistic methods; including ChIP-exo, growth phenotyping, conserved gene clustering, and transcriptome analysis. Among 47 LysR-type TFs (LTFs) found on the E. coli K-12 genome, many regulate nitrogen source utilization or amino acid metabolism. However, 19 LTFs remain unknown. In this study, we elucidated the regulation of seven of these 19 LTFs: YbdO, YbeF, YcaN, YbhD, YgfI, YiaU, YneJ. We show that: (1) YbdO (tentatively re-named CitR) regulation has an effect on bacterial growth at low pH with citrate supplementation. CitR is a repressor of the ybdNM operon and is implicated in the regulation of citrate lyase genes (citCDEFG); (2) YgfI (tentatively re-named DhfA) activates the dhaKLM operon that encodes the phosphotransferase system, DhfA is involved in formate, glycerol and dihydroxyacetone utilization; (3) YiaU (tentatively re-named LpsR) regulates the yiaT gene encoding an outer membrane protein, and waaPSBOJYZU operon is also important in determining cell density at the stationary phase and resistance to oxacillin microaerobically; (4) YneJ, re-named here as PtrR, directly regulates the expression of the succinate-semialdehyde dehydrogenase, Sad , and is a predicted regulator of fnrS . PtrR is important for bacterial growth in the presence of l-glutamate and putrescine as nitrogen/energy sources; and (5) YbhD and YcaN regulate adjacent y-genes on the genome. We have thus established the functions for four LTFs and identified the target genes for three LTFs.Although  The predicted yTF targets annotated as transporters and enzymes define the TF physiological role. A combination of the knowledge from EcoCyc, Fitness Browser, and iModulonDB with TF DNA-binding data provided hypotheses for the putative physiological functions of yTFs under different growth conditions2. Indeed, as an example, the function of one TF, PunR, was recently discovered to be an activator for punC, purine transporter important for adenosine utilization as a nitrogen source. Recently novel regulators for csgD were found by the promoter-specific screening of the 198 purified TFs including yTFs: YbiH, YdcI, YhjC, YiaU, YjgJ and YjiR. Two regulators for csgD: repressor YiaJ and activator YhjC (renamed RcdB) were found3. The SELEX method for the detection of the YbiH DNA-binding sites and antibiotics sensitivity for the yTF deletion mutant were used to predict the function for the CecR(YbiH)4.Recently, the roles of uncharacterized LysR-type transcription factors (LTFs) have been identified via multiple approaches, including transcriptome analysis of uncharacterized TF (yTF)-deleted mutants (machine-learning-based), gene clustering, and the detection of DNA-binding sitesEscherichia coli, LTFs are regulators for amino acids (AAs), purine, and dicarboxylate metabolism, nitrogen assimilation (NAC), antibiotic resistance, and virulence. LTF regulatory proteins protect 50–60 bp regions with TA-rich regulatory binding sites and activation binding sites (ABS) and regulate different metabolic pathways. LTFs are known to regulate conserved gene clusters that are adjacent to the genes encoding the regulator5, but additionally, LTF autoregulation is a common property. As one of the largest families of HTH-type regulators, LTFs contain an N-terminal helix-turn-helix DNA-binding domain and C-terminal co-inducer binding domain  have known functions and the majority were shown to regulate adjacent genes7. However, there are still many uncharacterized TFs belonging to the LysR-type family, which require further studies to determine their regulatory functions. This effort is also important for the reconstruction of the transcriptional regulatory network (TRN). The set of the six yTFs from LysR family  upregulated in the presence of l-threonine as supplement  to minimal growth medium and YneJ, transcriptional analysis for the regulation of putrescine/l-glutamate utilization in minimal medium were characterized in our research.LTFs account for 16.7% (47 out of 280) of the total number of transcription factors in Escherichia coli is a representative of the commensal mammalian intestinal microbiota and is the best characterized model gram-negative bacterium. Nutrient starvation conditions are important for the gut microbiome bacterial community as they cause stress, activating different survival mechanisms8, and TRNs rewire the metabolism under different conditions. iModulonDB is a collection of E. coli MG1655 transcriptomics data for different growth medium and stress conditions9. Machine-learning based ICA data analysis (iModulonDB), RNA-seq data for the TF knockout, and ChIP-exo data (in vivo DNA-binding sites) are useful resources for LTF characterization. Recently, the ChIP-exo results for verified uncharacterized TFs in E. coli MG1655 was published1. The gene expression profiling for LTFs under multiple growth conditions in iModulonDB provides important information for predicting the growth conditions to verification of function for the yTFs9.ybdO, ybeF, ycaN, ybhD, ygfI, yiaU, and yneJ), analyzed the conserved genome clustering with LTF genes, and detected conserved genes that were differentially expressed in the LTF knockout. Accordingly, we generated a hypothesis about the possible regulatory targets of three LTFs and their physiological functions in response to the high l-threonine concentration in minimal medium  detected LTF DNA-binding sites, transcriptome data from iModulonDB Fig. , RNA-seqium Fig. . The LTFium Fig.  had beensad gene expression in response to GABA, an intermediate of the Ptr utilization pathway.Here, using a transcriptomic analysis systems approach, we identify the LTFs, YbdO, YgfI, YcaN, YbhD, important for Thr utilization pathway in minimal medium and regulating catabolism of citrate, pyruvate, formate or malate, and YiaU, YbeF important for liposaccharide modification and flagella biosynthesis and YneJ (PtrR), that directly controls yneJ-sad cluster is conserved in enterobacterial genomes, and Sad is involved in putrescine utilization as nitrogen source10. Many bacterial species, including E. coli, can simultaneously utilize l-glutamate (Glu) and the polyamine putrescine (Ptr) under carbon/nitrogen starvation conditions. Glu is also essential for tetrahydrofolate polyglutamylation11. Ptr is important for bacterial growth and for efficient DNA replication, transcription, and translation13 and plays an important role in maintaining compact conformations of negatively charged nucleic acids14. Ptr is also involved in multiple antibiotic resistance mechanisms under stress conditions15. The puuA, puuD, puuE, and puuP genes in E. coli are induced by Ptr and regulated at the transcriptional level by the Ptr-responsive repressor PuuR17. The expression of sad (yneI) is induced by the addition of Ptr to the medium18; however, it is not regulated by PuuR, and a transcriptional regulator for sad had not been described before this work.For the detailed analysis, we investigated the effect of the YneJ transcriptional response in minimal medium under nitrogen starvation conditions. The sad promoter region was confirmed via an in vitro binding assay with the purified PtrR protein and using the ChIP-exo assay. We further compared the whole genome transcriptional response of the ptrR knockout and wild type E. coli strains to carbon/nitrogen starvation in the presence of Ptr and l-glutamate using RNA-Seq analyses, and the PtrR DNA-binding site was predicted for fnrS, encoding small regulatory RNA. The physiological roles of PtrR regulation and antibiotic resistance are discussed.The predicted PtrR binding site in the 19. Among these TFs, it was predicted that YeiE, YfeR, YidZ, YafC, YahB, YbdO, YbeF, YbhD, YcaN, YdcI, YdhB, YeeY, YfiE, YgfI, YhaJ, YhjC, YiaU, YneJ, and YnfL belong to the LTF based on Hidden Markov Model. YdhB (re-named PunR) function had been shown to be an activator for the purine transporter, PunC2. Recently YdcI  function was shown related to biofilm formation20. We further chose 7 uncharacterized LysR-type TFs (yLTFs), which have transcriptional responses (increased mRNAs) to the presence of a l-threonine (Thr) in M9 medium . l-threonine is an important source for l-serine, l-glycine, branched-chain amino acid biosynthesis, and formate, which is important for anaerobic respiration. To elucidate roles of each yLTF and genome-wide target genes, we performed gene expression profiling via RNA-Seq and ChIP-exo detection of the LTF DNA-binding and growth phenotyping. The overall workflow is shown in Fig. Previously, we generated a list of candidate transcriptional factors (TFs) from uncharacterized genes (“y-genes”) using a homology-based algorithmcitR-ybdNM  and ybdNM operons are conserved adjacent genes in Enterobacteriaceae . Further, gene expression profiling showed that citR deletion leads to strong upregulation of the ybdNM operon and the adjacent genes citCDEF. Interestingly that genome context analysis evolved that citR is conserved with the citrate lyase encoding genes cluster citCDEF.  and probably related to cit operon regulation. The DNA-binding site upstream of the citR gene was predicted by the phylogenetic footprinting method  ChIP-exo result detected the peak for DNA-binding upstream of dNM Fig. A,B, thateae Fig. D and theEF. Fig. C. Therefhod Fig. , suggestcitEFG), the phenotype for growth in the M9 medium supplemented with citrate at low pH (pH 6.5) showed an effect of citR deletion on citrate utilization, suggesting artificial de-repression of citrate lyase  and microaerobic utilization of formate as carbon source . The FlhDC iModulon includes FlhDC regulated genes that were upregulated in the ybeF and citR deletion mutants , presented in conserved gene clusters in Enterobacterial genomes with citrate lyase operon nts Fig. . LrhA ishDC Fig.  was subsybhD and ycaN mutant strains and WT were collected at the late-exponential phase after growth in the M9 medium supplemented with Thr. The conserved gene cluster ycaN-ycaK-ycaM with adjacent ycaC and ycaD genes was detected in the Escherichia coli K12 and Shigella boydii genomes. We noticed that in the ycaN deletion mutant strain differentially expressed genes ycaC (downregulated) and ycaD, focA (encoding formate channel) (upregulated) are genes adjacent to ycaN. It is interesting that ycaK, ycaC, and ycaD are predicted to be regulated by Nac (belonging to LTF), as Nac function is nitrogen assimilation, and ycaC and ycaD are probably nitrogen assimilation function related (EcoCyc). In ycaN deletion mutant highly DEGs are the tnaAB genes, encoding l-tryptophanase and a tryptophan transporter, l-arginine degradation, astCADBE, an autoinducer-2 transport system , and the HTH-type transcriptional regulator, galS, the genes were strongly downregulated (Supplementary Data 1). The genes encoding amino acid metabolism , threonine dehydrogenase (Tdh), transcriptional activator (TdcA), and glycolate utilization pathway ) were additionally downregulated in the ycaN mutant. The ycaN deletion mutant phenotype microarray applied for 95 carbon sources had evolved the negative phenotype for formate utilization specifically suggesting YcaN dependent focA regulation involved formate utilization  and the functional relation to nitrogen assimilation is suggested. The ybhD deletion strain growth phenotype in the M9 minimal medium with glycerol (carbon source), supplemented by l-malate was detected  . Accordingly, we decided to test the effect of the dhfA deletion on the growth on glucose , fumarate reductase (frdABCD), formate hydrogenlyase , and the regulator (hycA), as well as hydrogenase encoding genes  and the gene encoding protein involved in maturation of all hydrogenases isozymes (hypB)  Fig. A-B. The han Fig. B  Fig. F and therce Fig. C and witrce Fig. D aerobicium Fig. .Figure 3dhaKLM and the defect for the growth on glycerol  biosynthesis glycero-d-manno-heptose kinase) . Additionally, ChIP-exo detected binding for the genes encoding adenine transporter, adeP, suggests transcriptional regulation, and they were detected as DEGs for the yiaU mutant  in the M9 glucose medium. The phenotype (log phase) for the mutant in M9 with 0.3 M NaCl added was additionally detected  Fig. A. The rese) Fig. B. Accordor Table ; previouant Fig. D. We suglly Fig. C in RPMIted Fig. D, althouted Fig. D.Figure lpsR-yiaT are conserved in the bacterial genomes  interacting protein.The LysR family representatives are known to regulate adjacent genes, and mes Fig. E. The RNE. coli  via γ-glutamylated intermediates. The alternative pathway of Ptr degradation to GABA consists of PatA (Ptr aminotransferase) and PatD 24. The PuuABCDE pathway is essential for Ptr utilization in E. coli using PuuP as the major Ptr transporter25. GABA is further utilized by two alternative 4-aminobutyrate aminotransferases (GABA-AT) encoded by gabT and puuE, and also two succinate semialdehyde dehydrogenases (SSADH) encoded by gabD and sad27.Two distinct Ptr utilization pathways are known for oli Fig. A. The fi1. The ptrR gene is located in a conserved gene cluster with the divergently transcribed sad (yneI) gene, which encodes succinate semialdehyde dehydrogenase and yneH (glsB) glutaminase 28. To identify and characterize DNA binding sites of PtrR in the E. coli genome we utilized a combined bioinformatics and experimental approach. First, we applied a comparative genomic approach of phylogenetic footprinting27 to predict putative PtrR-binding sites in the common intergenic region of the ptrR and sad genes   contains a conserved 15-bp palindromic sequence with consensus TTCACnAATnGAGAA downstream predicted sigma-E dependent promoter , where the sad gene ortholog is absent and ptrR is co-localized with an uncharacterized MFS-family transporter gene. Further, we predicted two conserved DNA sites with similar consensus sequences located in their common intergenic region  by analyzing the PtrR ChIP-exo detected binding sitesase Fig. A and 6C,nes Fig. . The ptrsad genes in E. coli and conducted genome-wide mapping of other PtrR-binding sites using the ChIP-exo method. To identify in vivo PtrR binding sites, E. coli was grown under glucose as the carbon source in the M9 minimal media. A total of nine PtrR-binding sites were detected in these experiments. PtrR binds in the promoter regions of the fhuC, moeB, dhaK, fnrS,  gltS/xanP, and ptrR/sad genes. The experimentally identified 50-bp PtrR-binding region at sad/ptrR genes contains the conserved palindromic DNA motif identified via phylogenetic footprinting , and fnrS, respectively.We further confirmed the identified putative PtrR-binding site upstream of the ing Fig. . Comparisad promoter region has been facilitated using a PtrR-binding fluorescent polarization (FP) assay   and ptrR mutant strain growth phenotype on glutamate as the nitrogen source in minimal medium (glycerol as the carbon source) has been detected for the growth. Cells showed a growth phenotype when the ptrR gene was deleted under the starvation conditions  null mutant strains are shown in Fig. E. coli WT strain had a longer lag-phase compared to the ptrR mutant. A growth deficiency for a glsB mutant was observed under the same conditions, suggesting a functional relationship between GlsB (YneH) and Sad, encoding genes conserved in genome clusters with ptrR. The effect of a yneH deletion was substantial as cells approached the stationary phase.The phenotype microarray using Biolog PM2A was tested under the microaerobic conditions for the ent Fig. . The regE. coli WT and ptrR mutant were grown aerobically in M9 medium with 20 mM Glu and Ptr as nitrogen sources and 0.2% glycerol. To determine the effect of the ptrR deletion mutation, the cells were collected at the log-phase, and total mRNA was purified . PuuR and PuuADE, SodB, and two copper related transport systems’ mRNA levels increased in the ptrR mutant strain   Fig. . Howeverl-threonine for all yTFs, except PtrR. The transcriptional analysis for the ptrR deletion mutant was detected in the M9 medium with Ptr and/or Glu as nitrogen source. For LTFs, conserved adjacent genes had been shown to be differentially expressed. For example, gene clusters encoding ybdNM and citrate utilization genes citCDEF (citrate lyase) were detected as transcriptionally regulated in the citR deletion mutant. The CitR DNA-binding site upstream of citR has been predicted and confirmed by ChIP-exo assay, suggesting autoregulation. CitR has been shown to be important for the growth in minimal medium supplemented by citrate at acidic conditions, suggesting citrate lyase regulation microaerobically. Additionally, flagella biosynthesis genes (FlhDC regulon) were differentially expressed in the mutant. The citR mutant in E. coli BW25113 fitness phenotype had been previously shown for motility in LB medium. Additionally, CitR is important for E. coli BW25113 fitness in minimal medium with glycolate as the carbon source (fit.genomics.lbl.gov), and D-glycine as the nitrogen source. The phenotype for formate and l-glutamate utilization citR deletion mutant was detected microaerobically , YbeF, YbhD, YcaN, YiaU (LpsR), YgfI (DhfA), and YneJ (PtrR) Table . The trally Fig. . YbeF isdhaKLM operon for dihydroxyacetone phosphotransferase was detected by ChIP-exo. The transcriptome analysis shows that DhfA was important for regulation of dhaKLM, pflB, hycBCDEFG, narZYWV and adhB. The dhfA mutant growth phenotype using glycerol as a carbon source had been detected  DNA-binding site upstream of the ted Fig. C. The coal Table . The Pflrce Fig. F. The dhype Fig. , suggestwaaPSBOJYZU operon gltB and adeP genes, suggesting LpsR relationship to glutamate metabolism. lpsR deletion mutant transcriptomic analysis suggested the regulation of yiaT, the conserved adjacent gene to lpsR, which is divergently transcribed and yeaK, encoding deacylase for mischarged aminoacyl-tRNA   Table , Fig. 4DybhD adjacent gene ybhH conserved in enterobacterial genomes was upregulated in the ybhD deletion mutant. The ybhH, ybhI, and ybhD genes are conserved adjacent genes and potentially regulated by Nac (EcoCyc). YbhI is the putative tricarboxylate transporter, homologous to 2-oxoglutarate/malate translocator, (id. 35%) , supplemented by l-malate  Fig. 32. We deate Fig. , but noton Table ., FnrS. PtrR was predicted to be a repressor of the fnrS gene, encoding a small regulatory RNA  and phenotype microarray data .We detected that PtrR (YneJ) is the transcriptional regulator for Sad and the small RNANA Table . We demosad gene under nitrogen/carbon starvation has been investigated and discussed. The known stress/starvation sigma σS-controlled csiD-ygaF-gabDTP region is related to GABA utilization, while Sad is important for Ptr utilization35.PtrR-mediated regulation appears to be important for Ptr utilization as an energy source. A pleiotropic effect of the PtrR-dependent regulation of the 36. Our results demonstrated that PtrR is important for the growth of the E. coli BW25113 strain with Glu as the sole nitrogen source and glycerol as the carbon source and resistance to the tetracycline group of antibiotics , but not to chloramphenicol, erythromycin, and other antibiotics. PtrR is potentially important for the regulation of the highly conserved, anaerobically induced small RNA- fnrS, which is likely important for regulation under anaerobic growth conditions37. Interestingly, a ptrR mutant was shown previously to be resistant to bacteriophage lambda infection38 and we found that PtrR is potentially related to tetracycline resistance39. ChIP-exo and RNAseq results have been analyzed, providing a hypothesis for the physiological functions of YneJ , YgfI , and YbdO .Extracellular Ptr alters the OmpF porin charge and pore size, resulting in partial pore closure and a consequent decrease in outer membrane permeability40. LTFs are not always expressed under laboratory growth conditions .The identification of the additional DNA-binding sites for YgfI, YcaN, YiaU, YbeF, YneJ by gSELEX (genomic SELEX) method in the presence of Thr in the minimal medium possibly will provide additional information about novel LTFs transcriptional regulatory networkE. coli BW25113 and MG1655 strains transcriptomic data, ChIP-exo DNA-binding data for LTF, and the regulated biochemical pathway reconstruction and fitness/phenotype of the LTF deletion mutants strains produce fruitful hypotheses for the yTF function prediction that is important for TRN reconstruction in E. coli.Taken together, the systems analysis of the ptrR mutant strain. Pre-cultures were obtained by scraping frozen stocks and growing the cells in LB medium. Cells were washed twice with M9 medium and inoculated to an OD600 of 0.05. The cells were collected at an OD600 of 0.9  and were harvested using the Qiagen RNA-protect bacteria reagent according to the manufacturer’s specifications. Pelleted cells were stored at − 80 °C, and after cell resuspension and partial lysis, they were ruptured with a beat beater; the total RNA was extracted using a Qiagen RNA purification kit. After total RNA extraction and subsequent ribosomal RNA removal, the quality was assessed using an Aglient Bioanalyser using an RNA 6000 kit. The data processing is described in Supplemental materials.The wild type BW25113 strain was grown as a control for the isogenic 600 of 0.6 was reached. The cells were harvested after 4 h and lysed, the cell pellet was resuspended in the lysis buffer. Rapid purification of recombinant proteins on Ni-nitrilotriacetic acid-agarose minicolumns was performed. The protein was refolded on a mini-column, and the buffer was changed to a buffer containing 0.1 M Tris–HCl, 0.1 M NaCl, 10 mM urea.The PrtR-producing strain was grown overnight, re-inoculated into 50 mL of the fresh medium, and induced with 0.6 mM IPTG after an OD4, 5 mg/ml sperm DNA and 1uM of the fluorescently labeled predicted PtrR binding DNA fragment as well as 0–0.6 mM GABA. Then the PtrR protein (0–1.5 uM) was added to the assay mixture, and it was incubated for 1 h at 30 °C in the presence or the absence of GABA.The purified PtrR protein and 10 nM fluorescently labeled DNA fragment (5′-gggTTCTCGATTCGTGAAggg-3′) were incubated in the assay mixture. The PtrR binding assay mixture (0.1 ml) contained Tris buffer, pH 7.5, 0.1 M NaCl, 10 mM MgSO2SO4). The flow rate was held at 0.5 mL/minute, the sample injection volume was 10 uL, and the column temperature was maintained at 45 °C. The identities of compounds were determined by retention time comparison to standard curves of acetate, ethanol, glucose, lactate, pyruvate, formate and succinate. The peak area integration and resulting chromatograms were generated within ChemStation and compared to that of the standard curves to determine the concentration of each compound in the samples.For organic acid and carbohydrate detection, samples were collected after 4 h for every 30–45 min. The filtered samples were loaded onto a 1260 Infinity series (Agilent Technologies) high-performance liquid chromatography (HPLC) system with an Aminex HPX-87H column (Bio-Rad Laboratories) and a refractive index detector and HPLC was operated using ChemStation software. The HPLC was run with a single mobile phase composed of HPLC grade water buffered with 5 mM sulfuric acid  or RPMI with 10% LB (RPMI_LB) for PM12B and inoculated as recommended to the Omnolog plates PM1, PM2, PM11C or PM12B, for the antibiotic resistance measurements at 37 °C. The experiments were repeated two times.The Supplementary Information 1.Supplementary Information 2.


The lymphatic system is crucial for the maintenance of interstitial fluid and protein homeostasis. It has important roles in collecting excess plasma and interstitial fluid leaked from blood vessels, lipid absorption and transportation in the digestive system, and immune surveillance and response. The development of lymphatic vessels begins during fetal life as lymphatic endothelial progenitor cells first differentiate into lymphatic endothelial cells (LECs) by expressing the master lymphatic vascular regulator, prospero-related homeobox 1 (PROX1). The lymphatic vasculature forms a hierarchical network that consists of blind-ended and unidirectional vessels. Although much progress has been made in the elucidation of the cellular and molecular mechanisms underlying the formation of the lymphatic vascular system, the causes of lymphatic vessel abnormalities and disease are poorly understood and complicated; specifically, the mechanistic basis for transcriptional dysregulation in lymphatic vessel development remains largely unclear. In this review, we discuss the recent advances in our understanding of the molecular and cellular mechanisms of lymphatic vascular development, including LEC differentiation, lymphangiogenesis, and valve formation, and the significance of mechanical forces in lymphatic vessels, with a focus on transcriptional regulation. We also summarize the current knowledge on epigenetic mechanisms of lymphatic gene expression. In this review, we provide an update on the current knowledge regarding the development of the lymphatic vasculature and its mechanical force signals, especially focusing on transcriptional regulatory mechanisms.A well-organized lymphatic system including proper lymph fluid absorption and drainage is imperative for maintaining interstitial fluid and protein homeostasis. The lymphatic system is composed of a blind-ended, unidirectional network that contains absorptive vessels, primary lymphoid organs such as thymus and bone marrow, secondary lymphoid organs such as lymph nodes, spleen, and Peyer’s patches, and lymphoid tissues as adenoids and tonsils . LymphatPROX1 positive lymphatic endothelial progenitor cells expressing vascular endothelial growth factor receptor (VEGFR) 3 sprout via stimulation with mesenchyme-derived VEGF-C ligand. These cells further migrate dorsolaterally from cardinal and intersomitic veins and establish primary lymph sacs and superficial lymphatic vessels identified as the jugular lymph sac by E11.5 ; therefore, they work for collecting excess plasma and interstitial fluid leaked from blood vessels . In cont process . VEGFR3 or cells . In collor cells .PROX1 in venous endothelial cells (VECs), leading to the misconnection of blood and lymphatic vessels  4, and SRY-box (SOX) 17. Of note, mosaic overexpression of SOX17 in zebrafish ECs results in reduced lymphatic gene expression in the anal fin as well as the absence or incomplete formation of the thoracic duct , which is involved in the differentiation of various cell types, regulates the transcriptional changes invoked by VEGF-C in LECs . MAFB inhenotype . LEC-speogenesis . The traelopment .FOX O1 in mice decreases LEC migration toward CXCL12 by downregulating C-X-C chemokine receptor (CXCR) 4, induces excess LEC proliferation, and decreases LEC apoptosis, which leads to the disconnected and dilated structure of the lymphatic vessels (Forkhead box (Fox) O1, a member of the Fox transcription factor family, acts an essential role in developmental lymphangiogenesis by promoting LEC migration toward the chemokine (C-X-C motif) ligand (CXCL) 12 and regulating their proliferative activity . The LEC vessels .COUP-TFII promoter and impacting the ability of transcriptional machinery to access the promoter (Brg1 in mice results in downregulation of COUP-TFII expression in developing veins (Brahma-related gene 1 (BRG1), a chromatin-remodeling enzyme, epigenetically regulates COUP-TFII expression, by remodeling chromatin within the promoter . The EC-ng veins .CHD4 controls the development of lymphovenous valves, which regulates the return of lymph to the blood circulation by forming fibrin-rich thrombi that prevent blood from entering the lymphatic system (CHD4 in mice leads to increased transcription of the urokinase plasminogen activator receptor (uPAR), thereby facilitating activation of the fibrin-degrading protease plasmin and then degrading the fibrin near the lymphovenous valves , an ATPase subunit of the nucleosome remodeling deacetylase (NuRD) chromatin-remodeling complex, regulates vascular integrity in the mid-gestation . Specific system . The LECs valves . In addis valves . The Hips valves . LEC-speprouting .DOT1L causes fully penetrant lymphatic aplasia by altering the lymphatic transcription program and reducing H3K79me2 enrichment at lymphatic genes, including the transcription factors SOX18, SOX17, and FOXC2, which are critical for LEC differentiation and valve formation, as well as Vegfr3, which is critical for LEC proliferation and migration (Disruptor of telomeric silencing 1-like (DOT1L), a histone H3 lysine (H3K) 79 methyltransferase, promotes transcription by histone methylation of chromatin and is a crucial factor in the homeostasis of various organs such as the heart and hematopoiesis . Vasculaigration   1A expression, which increases fatty acid β-oxidation-dependent acetyl coenzyme A production; 2) Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes; 3) histone acetyltransferase p300 interacts with Prox1 to facilitate preferential histone acetylation at the loci of PROX1-targeted genes  PROXed genes . LEC-spet E16.5. . Other tpromoter . In addith PROX1 . Additioromoters .PROX1 and FOXC2 expression, whereas Foxc2 regulates valve maturation in cooperation with Prox1 to control intraluminal invagination of LECs and reorganization into valve forming leaflets by postnatal day (P)1 1 . As an uday (P)1  1 .FOXC2 is a causative gene whose mutations are dominantly associated with lymphedema-distichiasis syndrome characterized by failure of lymph drainage in limbs, venous valve failure, and the growth of an extra set of eyelashes  is significantly impaired , a cation channel activated by mechanical forces such as fluid shear stress or membrane stretch, is a causative gene associated with congenital lymphedema with pleural effusion . The LECant mice . Anothernd GATA2 . Moreove profile . Treatmet manner . TogethePROX1 and VEGFR3 have started to clarify LEC heterogeneity in various organs including functional multiformity. For example, single-cell RNA-sequencing analysis using the zebrafish anal fin was the key to characterizing the different endothelial cell populations and transition states involved in the LEC transdifferentiation process (Many studies have been conducted on the formation, maintenance, and function of lymphatic vessels, which are essential to maintain homeostasis. This review focuses on the mechanisms of transcriptional regulation in LECs during lymphatic vessel development , but the process . Moreove process . Yet, ad


Saccharomyces cerevisiae, TI is mainly driven by antisense non-coding transcription and occurs through re-shaping of promoter Nucleosome-Depleted Regions (NDRs). In this study, we developed a genetic screen to identify new players involved in Antisense-Mediated Transcription Interference (AMTI). Among the candidates, we found the HIR histone chaperone complex known to be involved in de novo histone deposition. Using genome-wide approaches, we reveal that HIR-dependent histone deposition represses the promoters of SAGA-dependent genes via antisense non-coding transcription. However, while antisense transcription is enriched at promoters of SAGA-dependent genes, this feature is not sufficient to define the mode of gene regulation. We further show that the balance between HIR-dependent nucleosome incorporation and transcription factor binding at promoters directs transcription into a SAGA- or TFIID-dependent regulation. This study sheds light on a new connection between antisense non-coding transcription and the nature of coding transcription initiation.Eukaryotic genomes are pervasively transcribed by RNA polymerase II (RNAPII), and transcription of long non-coding RNAs often overlaps with coding gene promoters. This might lead to coding gene repression in a process named Transcription Interference (TI). In   Maps ofin tandem or in antisense configurations with respect to the coding gene. High levels of nascent antisense transcription into promoters in Eukaryotes correlate with significantly lower levels of coding sense transcription . pGAL-H3-3xHA-URA3 on a single copy plasmid is a gift from the Michel Strubin lab.TDH3-dCas9-LEU2 was obtained from Addgene (Plasmid #46920)  using OFS2869 and 2870 (-TRP1 from Addgene (Plasmid #49014) and OFS2871 and 2872 to amplify the insert (URA3 with its promoter) from YCpLac33 plasmid backbone. The plasmid PHO84 AS gDNA-URA3 was then obtained by Gibson assembly reaction (NEB) using OFS2886 and 2887 to amplify Empty-gDNA-URA3 backbone and OFS2888 and 3017 for gDNA cloning.The plasmid p #46920) . The plaand 2870  to ampliPHO84 gene respectively, left to grow for 2 days at 25°C and then transferred to sporulation medium at 22°C for 7 days. Finally, Clon-NAT-pho84Δ::HIS3 rrp6Δ::LEU2 DEL::Kanr haploids were sequentially selected on adequate selective synthetic medium over a total duration of 7 days at 25°C before being assessed for enhanced growth on His– plates.The detailed synthetic genetic array screening procedure is described in ,41. FresFlow cytometry was perfomed as described in .2O and 2 μl were used for amplification by qPCR. Primers used for reverse transcription and PCR are listed in RNA was extracted using Glass-beads and TRIzol (Invitrogen). 10 μg of total RNA was further treated with TURBO DNase (Invitrogen). Then, 67 ng of total RNA was reverse transcribed using SuperScriptII (Invitrogen) in a 20 μl reaction containing dNTPs , sense or antisense oligos for a given target gene  and SCR1 antisense primer . Reverse-transcription proceeded for 50 min at 42°C before 15 min of incubation at 70°C. The 20 μl reaction was then diluted 5 times in HThe MNase-seq experiments were performed as described in . LibrariThe experiment was performed as described in  except t600= 0.2 to resume exponential growth until OD600= 0.4. α-factor  was added to the culture for 3 h at 30°C to synchronize cells in G1-phase. Galactose powder  was then directly added to the medium to induce H3HA synthesis and the culture was split to add or not rapamycin  for 1h. Cells were then processed for MNase-ChIP-seq as in .The Nrd1-AA strain transformed with the pGAL-H3-3xHA-URA3 plasmid (gift from Michel Strubin lab) was grown overnight in SC -URA + 2% Raffinose at 30°C. Cells were then diluted to ODeq as in . Newly s(–Ct).The experiments were performed as described in , withoutSaccharomyces Genome Database (SGD) giving a complete list of 4775 coding genes. For the TBS coordinates, our list was crossed with the ChIP-exo data from the Pugh lab  to poly-A was kindly provided by the Mellor Lab. Among them were picked the ones considered as ‘Verified’ genes in the Pugh lab . +1 nuclPugh lab  and fromPugh lab . FiguresPugh lab . The lisPugh lab  and crosPugh lab . Reb1 rePugh lab  via analned from .Saccharomyces_cerevisiae.EF4.65.gtf). Antisense transcripts with a fold-change of at least two and multiple testing adjusted P-value lower than 0.05 were considered differentially expressed and defined as inducible Antisense Genes (iAS). Calculations of fold-changes were subsequently performed using the density files (bigWig files).Single-end reads were aligned to sacCer3 genome assembly using Bowtie2  with optPaired-end reads were aligned to sacCer3 genome assembly using Bowtie2 . PCR dupAdapters were first removed from the paired-end reads using the Trim Galore! Tool with default options from the Galaxy server . Paired-Metagene plots were produced using computeMatrix followed by plotProfile commands using DeepTools 2.0 . HeatmapU tests were used to extract a P-value. ns if P-value > 0.05, ∗ < 0.05, ∗∗ < 0.01, ∗∗∗ < 0.001, ∗∗∗∗ < 0.0001. All models were designed using BioRender.All plots and statistical analyses of this work were performed using Prism 8.0 (Graphpad). All tests are nonpaired tests. Mann–Whitney PHO84 model gene for which coding sense expression is regulated by antisense non-coding transcription. PHO84 becomes repressed in the absence of Rrp6, the nuclear subunit of the exosome, as a result of decreased Nrd1- and Nab3-dependent early termination of antisense transcription and subsequent invasion of the sense promoter by antisense transcription  approach in which a catalytically dead Cas9 protein (dCas9) is directed by a guide RNA (gRNA) to block transcription in a strand-specific manner  > 0, defined as the first criterion) >0), defined as the second criterion)  at the genome-wide level, we performed RNA-seq of an Nrd1 anchor-away strain (Nrd1-AA), in which Nrd1 early terminated antisense non-coding transcription can be extended into gene promoters upon rapamycin addition ,54. This Figures . Moreove Figures . In the  Figures . We then Figures . These 6 Figures . The gen Figures . As alre Figures , iAS gen Figures .HIR2 does not lead to AMTI alleviation following the first and second criteria when iAS genes are taken as a whole  of the Hir2 subunit , we, we16),  Figures  and C.HA incorporation at promoters upon antisense extension is Hir2-dependent -Binding Site (TBS) for the iAS as compared to the niAS Figures –F. Moreot Figure . Increasncreases . Thus, increases .HA incorporation at promoters is strictly dependent on increased antisense transcription and not on the SAGA- or TFIID-dependent nature of genes.Altogether, our results indicate a positive correlation between coding/non-coding transcription, HIR binding and replication independent H3 incorporation. However, it is worth noting that increased recruitment of Hir2 and H3S. cerevisiae coding genes are influenced by steady-state antisense transcription into promoters. Thus, some of the SAGA-dependent genes might have a high natural level of antisense that does not further increase upon Nrd1 anchor-away, and are therefore not classified as iAS genes. Thus, we decided to consider the whole set of SAGA-dependent genes in order to assess their natural antisense levels as well as their dependency on the HIR complex for coding gene expression.Only a small number of iAS genes are SAGA-dependent Figure  and E. HPHO84  present a significantly higher steady-state level of nascent antisense transcription into promoters as compared to TFIID-dominated genes 3998 genes)  a more closed chromatin structure ,28–30,  and . It is nevertheless quite consistent with the molecular model we propose. In order to be up-regulated, a SAGA-dependent gene has to lose a nucleosome masking the TF-binding site and the TBS. While the probability of this event is increased by high levels of antisense transcription elongation into promoters, it stays low since the recycling of already incorporated histones by essential histone chaperones is an efficient mechanism  ,68. We cS. cerevisiae (The typical chromatin organization of SAGA- and TFIID-dependent promoters appears as conserved in human cells and related to the transcriptional plasticity, like in yeast . Moreoverevisiae . Thus, mThe accession number for the data reported in this study is GEO: GSE175991.gkac264_Supplemental_FilesClick here for additional data file.