Escherichia coli cells. Using the polysome profiling method, the ribosome occupancy (RO) and ribosome density (RD) of different mRNA copies were determined for several hundred mRNAs during the exponential- and stationary-phases, providing the most complete characterization of such regulation in E. coli. Although for most genes, nearly all mRNAs (>90%) were undergoing translation, they were loaded with far fewer than the theoretical maximum number of ribosomes, suggesting translation limitation at the initiation step. Multiple linear regression was used to identify key intrinsic factors involved in the genome-wide regulation of RO and RD . Unexpectedly, mRNA concentration, a factor that depends on cell physiology, was predicted to positively regulate RO and RD during the exponential- and stationary-phases. Using a set of selected genes controlled by an inducible promoter, we confirmed that increasing the mRNA concentration upon transcription induction led to increases in both RO and ribosome load. The fact that this relationship between mRNA concentration and translation parameters was also effective when E. coli cells naturally adapted to carbon source changes demonstrates its physiological relevance. This work demonstrated that translation regulation is positively controlled by transcript availability. This new mechanism contributed to the codirectional regulation of transcription and translation with synergistic effects on gene expression and provided a systemic understanding of E. coli cell function.Translational regulation was investigated at the genome-scale in IMPORTANCE The process of gene expression is divided into translation and transcription. Considerable efforts have been made in bacteria to characterize the mechanisms underlying translational regulation and identify the regulatory factors for particular mRNAs. However, to understand bacterial physiology and adaptation, it is important to elucidate genome-wide translational regulation and examine its coordination with transcriptional regulation. Here, we provided a genome-wide picture of translational regulation in Escherichia coli. For most genes, nearly all mRNA copies were found to undergo translation but were loaded with a low number of ribosomes. We showed that mRNA concentration had a positive effect on translation regulation, linking translational regulation to transcriptional regulation as well as to cell physiology and growth conditions. The codirectional regulation of transcription and translation had synergistic effects on gene expression, contributing to E. coli cell function optimization. This finding could be used in biotechnology to optimize strategies for recombinant protein synthesis. Protein synthesis is an important cellular process because the translation of mRNA into proteins is the most energy-consuming process in bacterial cells . A funda2–E. coli has not been quantified.Translational regulation in bacteria has not been often studied but has been identified in response to environmental cues and stress –8, and e–11–E. coli but are required for a genome-wide understanding of translational regulation in this model bacterium.The development of high-throughput methods, such as ribosome and polysome profiling has made it possible to study translational regulation at the genome-wide level in bacteria. Genome-wide translational information is usually obtained in bacteria using the ribosome profiling method based on ribosome footprints , 16–20. 16–E. coli mRNAs in fast-and nongrowing cells. Multiple linear regression was used to identify and rank the effects of multiple parameters on the RO and RD of each mRNA. Strikingly, mRNA concentration, which depends on cell physiology and growth conditions, was predicted to be a significant regulatory factor of RO and RD in both growing and nongrowing E. coli cells. This positive effect of mRNA concentration on translation regulation identified at the omics scale was confirmed in a set of selected genes after induction of artificial transcription. Results for E. coli during adaptation to a new carbon source illustrate the physiological relevance of translational regulation by mRNA concentration.To determine the general rules of translational regulation and gain a better understanding of its coordination with the transcription process, we used polysome profiling to perform the first genome-wide analysis of the translational status of E. coli cells were cultured in the exponential- or stationary-phase (1 h after glucose depletion) to characterize the translational status of E. coli mRNAs in both phases  in exponentially growing cells  and 0.3 E. coli namely, the RBS score  were ranked. Although the coefficients of determination (R2) of the models were low (between 0.12 and 0.27), a large set of quantitative and qualitative factors involved in RO and RD regulation during the exponential- and stationary-phases was identified , the presence of the signal peptide, and being a target of CsrA were positively associated with RO only in one of the two culture conditions. In the exponential-phase, the length of the open reading frame (ORF) was a positive regulator of RO in contrast to the length of the 5’UTR.Regarding the regulation of RD, the GC% of the open reading frame (ORF) was strongly negatively associated with RD in both culture phases. An increase in the GC% of an ORF, most likely favoring secondary structure formation in the mRNA, was associated with a decrease in RD. This negative effect of sequence folding on RD was also observed in the translation initiation region (TIR) region. The codon adaptative index was positively associated with RD in the two culture conditions. mRNA concentration, protein hydrophobicity, and being a target of CsrA were positive factors of RD in one of the two culture conditions. Metabolic function (the clusters of orthologous groups [COG] parameter) was also associated with RD regulation in a culture phase-specific manner. In the stationary-phase, mRNAs related to amino acid and carbohydrate transport and those with unknown functions had lower and higher RDs, respectively, than those in other functional categories. Results related to protein localization were not so simple: membrane localization was positively associated with RD in the exponential-phase whereas being an inner membrane protein was negatively associated with RD in stationary-phase.These results identified the mRNA concentration as a factor regulating translation. Genes with high mRNA concentrations had significantly higher proportions of mRNA copies undergoing translation and higher numbers of the bound ribosome, as illustrated in lacZ gene by IPTG  induction and analyzed the distribution of lacZ mRNA copies in the different polysome profile fractions . As a result, the RO of the lacZ mRNA increased to 99% ± 1%.This positive correlation in the genome-wide models between mRNA concentration and RO and RD points toward a relationship between mRNA concentration and translational regulation. To validate this result experimentally, we increased the mRNA concentration of the native chromosomal ractions . WithoutcysZ, inaA, ucpA, yeeZ, yjcO, and lacZ) to changes in mRNA concentration by cloning these genes under the arabinose-inducible promoter. We first used the lacZ gene to confirm that the translational responses were similar using a chromosome and a plasmid . Upon induction with arabinose, the six corresponding mRNA concentrations were increased between 18- and 83-fold (Table S1). Polysome profiling was compared at low and high mRNA concentrations using parallel multiplexed experiments , and rpsJ and rplK mRNAs encoding ribosomal proteins were similar at high and low mRNA concentrations (Table S1) indicated that the observed association between high ribosome load and high mRNA concentration was not due to an increase in the concentration of free ribosomes.We investigated the generality of this result by analyzing the translational response of six genes . Under these conditions, lacZ mRNA copies shifted from fraction A (free) toward fractions F and G , indicating that translation initiation is efficient in E. coli. However, most mRNAs were loaded with a rather low number of ribosomes. Some studies found that the optimal RD to maximize the protein translation rate was significantly (50% to 80%) lower than the maximal theoretical density  and TIR region were associated with lower RD. This result is consistent with the negative correlation between per mRNA . The posper mRNA , 13. Regper mRNA . The binli cells , which iThe cellular localization of the protein was predicted to regulate translation: localization at the membrane was a negative factor of RO in both culture conditions, and positively/negatively associated with RD in the exponential- and stationary-phases. We do not know why membrane protein should be differentially translated and why it should depend on the physiological state. In the literature, controversial results on the link between translation and protein localization were reported confirming the difficulty to conclude on this point –32.–L. lactis  preventing the backtracking of RNA polymerase and enhancing transcription,  and/or  and, thuE. coli cells adapt their metabolism by adjusting gene expression. Here, we provide evidence of in vivo codirectional transcriptional and translational regulation of lacZ when E. coli transitions to growth on lactose. The high β-galactosidase activity required for growth on lactose was achieved through increases in the concentration and ribosome loading of lacZ mRNAs. From a physiological point of view  of 0.1 after overnight preculture. The partial pressure of oxygen was continuously monitored during the culture and maintained above 30%. Cells were collected in the exponential-phase at an OD600 of ∼1.3 and in the stationary-phase 1 h after glucose depletion at an OD600 of ∼3.4.For the genome-wide polysome profiling studies, lacZ , E. coli MG1655 was grown on M9 medium in a flask at 37°C at 150 rpm. For physiological lacZ overexpression, cultures were performed with 8.3 mM lactose as a carbon source, and cells were collected in the exponential-phase at an OD600 of ∼1.2. For artificial lacZ overexpression, cultures were grown on 3 g/liter glucose with 1 mM IPTG induction when cultures reached an OD600 of ∼1 for 30 min.For polysome profiling study of lacZ, we selected cysZ (sulfate transporter), inaA (putative lipopolysaccharide kinase), ucpA (putative NAD binding oxidoreductase), yeeZ (putative epimerase), and yjcO (unknown function). For each gene, the 5’UTR + ORF fragment was amplified by PCR and cloned in a PBAD/myc/His vector . Plasmids were introduced into E. coli MG1655 ΔaraFGH, Ωpcp18::araE533  obtained without arabinose induction, and one experiment on a mix of cell extracts of the six strains after arabinose induction.Strains were constructed to artificially increase the mRNA concentration of specific genes. In addition to :araE533 , except :araE533 . All str2, 0.5 mM DTT, 100 μg/mL chloramphenicol, 1 mg/mL heparin, 20 mM EGTA, 1% Triton X-100). Heparin was used as a RNase inhibitor. After mechanical cell disruption with glass beads, mRNA-ribosome complexes were size-separated on a sucrose gradient  into 24 subfractions. Total RNA was extracted using the RNeasy Midi kit  and quantified using a Nanodrop spectrophotometer . The levels of 16S and 23S rRNAs in each subfraction were calculated using the Bioanalyzer 2100  and used to pool the subfractions into seven fractions labeled A to G . Fraction A consisted of subfractions containing DNAs, free RNAs, and free small and large ribosomal subunits. In fractions B to G, the 23S/16S rRNA ratio was constant, at about 1.8, corresponding to entire ribosomes. The 1st peak (fraction B) was attributed to the monosome. The 2nd, 3rd, and 4th peaks were attributed to 2, 3, and 4 ribosomes, respectively. The number of ribosomes in the other fractions was extrapolated . Ribodepleted RNA (10 ng) was then used to construct a sequencing library using the Ion Total RNA-Seq kit v2 kit and the Ion Xpress™ RNA-Seq Barcode 1–16 kit . The library was constructed and (100 bp) single-end sequencing on an Ion Torrent system equipped with an Ion P1 Chip kit was performed by the GeT-BioPuces platform (http://get-biopuces.insa-toulouse.fr/). These experiments were repeated with three independent cultures. Reads were mapped onto the E. coli genome  using the Burrows-Wheeler alignment method (version 0.7.12-r1069) . rRNA was removed using the Ribo-Zero2-r1069) . Countini in fraction j and replicate k as follows:Finally, the variability in the initial amount of total RNA between fractions was considered, and a constant 5 μg of total RNA was used in each fraction for ribodepletion. We obtained the final normalized count for gene i, we calculated the proportion of mRNA copies in each fraction j for each replicate k:For each gene lacZ mRNA was quantified using the average value obtained from five primer pairs. In unfractionated samples, the concentration of target RNA was estimated as the relative RNA abundance compared to the constant quantity of ERCC spike-ins using the method of fold change ΔCt values  was reverse transcribed to yield cDNA using SuperScript II reverse transcriptase (Invitrogen) . cDNA wat values . In the t values .i, RO, the proportion of mRNA copies undergoing translation, was calculated as the total summed proportion of mRNAs bound to at least one ribosome (fractions B to G):RO and RD were determined for monocistronic genes using the RegulonDB database. For each gene The final RO for each gene was calculated as the mean RO measured in three replicates in the exponential- and stationary-phases.The peak fraction (the fraction with the highest proportion of mRNAs undergoing translation) was determined by the bootstrap method on residuals, with a confidence interval fixed at 95% . BrieflyicdC and ymgF) with outlier RDs (>3.3 ribosomes/100 nt) in the exponential-phase were excluded from subsequent analyses. The RD and RO values obtained in the exponential- and stationary-phases are shown in Table S4.Two genes  were obtained or calculated from GenBank (version 3). Gene essentiality and CsrA targets were obtained from Baba et al. , respectively. Genome-wide mRNA concentrations in the exponential- and stationary-phases measured in the same standardized growth conditions have been reported previously (P value was calculated for each parameter coefficient.The major regulatory factors for RO and RD were identified using multiple linear regression models with quantitative and qualitative parameters as predictor factors , 7, 8. Sa et al.  and Esqua et al. , respecta et al. . Parametnotation . Inner mnotation  and Signeviously , 37. Pareviously , 49. TheP values adjusted for multiple testing using the Benjamini and Hochberg false discovery rate method (Simple linear correlations between two parameters were estimated by calculating Spearman correlation coefficients and corresponding e method .Raw and processed RNA-seq data were deposited in the Gene Expression Omnibus data repository and are accessible through GEO accession GSE191073.


TET2. This review summarizes the latest molecular findings regarding TET2 functions in hematopoietic cells and their potential implications in blood cancer origin and evolution. Our goal was to encompass and interlink up-to-date discoveries of the convoluted TET2 functional network to provide a more precise overview of the leukemic burden of this protein.Bone marrow disorders such as leukemia and myelodysplastic syndromes are characterized by abnormal healthy blood cells production and function. Uncontrolled growth and impaired differentiation of white blood cells hinder the correct development of healthy cells in the bone marrow. One of the most frequent alterations that appear to initiate this deregulation and persist in leukemia patients are mutations in epigenetic regulators such as TET2 is frequently mutated in the hematopoietic system from the three TET family members. These mutations initially lead to the hematopoietic stem cells (HSCs) compartment expansion, eventually evolving to give rise to a wide range of blood malignancies. This review focuses on recent advances in characterizing the main TET2-mediated molecular mechanisms that activate aberrant transcriptional programs in blood cancer onset and development. In addition, we discuss some of the key outstanding questions in the field.Cytosine methylation (5mC) of CpG is the major epigenetic modification of mammalian DNA, playing essential roles during development and cancer. Although DNA methylation is generally associated with transcriptional repression, its role in gene regulation during cell fate decisions remains poorly understood. DNA demethylation can be either passive or active when initiated by TET dioxygenases. During active demethylation, transcription factors (TFs) recruit TET enzymes  to specific gene regulatory regions to first catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and subsequently to higher oxidized cytosine derivatives. Only  CpG methylation is the most common DNA modification found in the mammalian genome , playingTET2 is the most broadly expressed and frequently mutated gene in blood malignancies [TET2 mutational profiles show inactivating mutations occurring along the whole gene coding region and are not only restricted at the 3′ region where the catalytic domain is located [TET2 epigenetic regulation during blood differentiation might be mediated not only by its catalytic activity but also by its association with critical partners [In the context of the hematopoietic system, TET family members have been involved in naturally occurring and experimentally induced cell fate decisions ,12,13,14gnancies ,18,19,20 located ,17,21. Tpartners ,23.TET2 functions in normal and malignant hematopoiesis, providing an extensive overview of the intricate molecular mechanisms controlling gene expression, protein stability and function, and enzyme’s genome recruitment.Here we review the current understanding of TET2 expression and activity have been elucidated in the last decade. Here we summarize main control systems, encompassing basal post/transcriptional regulation, direct protein modulation through post-translation modifications (PTMs), and enzymatic substrate availability.Several mechanisms regulating Tet1 and Tet2 are positively regulated by the pluripotency TF Oct4 that binds to conserved non-coding sequences in both genes [Tet1 and Tet2 levels decrease due to Oct4 depletion [Some transcriptional factors have been defined as direct regulators of TETs’ gene expression. In mouse embryonic stem cells (ESCs), th genes . Accordiepletion .Tet1 and Tet2 expression [Tet2 expression is boosted by the action of the myeloid transcription factor CEBPα, which binds to Tet2 enhancer regions [Tet2 protein itself to Tet2 gene’s distal regulatory regions leading to their demethylation and activation [Tet2 enhancers [TET2 expression in the context of MDS and AML [Tet1 and Tet2 are regulated in response to hydrogen sulfide (H2S) through the action of the sulfhydrating nuclear transcription factor Y subunit beta (NFYB) [A similar regulatory mechanism was described for the CXXC-DNA binding domain protein Rinf (CXXC5), whose depletion leads to decreased pression . During  regions . CEBPα mtivation . Of notetivation , failed nhancers . In addi and AML . Finallya (NFYB) .TET2 mRNAs have been proposed as the primary post-transcriptionally regulatory mechanism during blood differentiation and in myeloid malignancies. High-throughput screens identified a large subset of TET2 3′UTR targeting miRNAs with different efficiencies. Induced expression of those led to an array of leukemic traits such as myeloid lineage bias, phenocopying to an extent, a direct TET2 loss [TET2 mRNA) was detected overexpressed in MDS patients. Mechanistically, miR-22 overexpression leads to reduced genome-wide levels of 5hmC, increased self-renewal, and myeloid skewing [TET2 by post-transcriptionally regulating SIRT1 levels, which affect the TET2 protein function at a post-translational level  recruits epletion . Similarepletion . Calpain in ESCs .TET2 protein can be largely post-translationally modified (PTM) by (de)phosphorylation, (de)acetylation, O-GlcNAcylation, or ubiquitylation, among others [TET2 activity is cell type and amino acid residue-specific. For instance, cytokine receptor-associated JAK2  phosphorylates TET2 at tyrosine residues 1939 and 1964, leading to enhanced enzymatic activity [TET2 tyrosine residues 1939 and 1964 are not the only phosphorylatable residues since the whole N-terminus region of the protein (constituting a large disordered domain) is usually highly phosphorylated in ESCs [In addition, g others ,36,37,38activity   leads to reduced enzymatic activity triggering the emergence of abnormal DNA methylation profiles typically associated with cancer [TET2 stability/activity mediated by histone acetyltransferase p300 (enzymatic counterpart of the HDAC1/2 enzymes). p300 was shown to acetylate the TET2 N-terminus region leading to increased protein activity, stability, and partnering with other proteins such as DNMT1 [TET2 activity can also be regulated through protein (de)acetylases. This is the case of the NAD-dependent deacetylase sirtuin-1 (SIRT1) that removes acetylation at activity . Consequpatients . Whereash cancer . Zhang aas DNMT1 . TET2/DNas DNMT1 .TET2 has been described to regulate its chromatin association [TET2 and promote K1299 monoubiquitylation, enhancing its chromatin association [TET2 activity [Finally, ubiquitylation of ociation . CRL4  and succinate dehydrogenase (SDH) lead to fumarate and succinate accumulation that inhibit Tet enzymatic activity even with stable α-KG levels [TET2 might also be directly relevant in clinics as mutations in iron and 2-oxoglutarate-binding sites have been reported in AML patients [TET2 expression and activity in leukemic cells through a mechanism involving the activation of the hypoxia-inducible factor 1α (HIF1α) [Regarding catalytic activity, TET enzymes are Fe2+/α-KG-dependent dioxygenases. Metabolite and cofactor availability constitute another relevant layer of protein regulation potentially influencing hematopoiesis and leukemic development. Interestingly, experimentally-induced ascorbate (VitC) depletion leads to increased HSC function and compartment expansion, resembling the aberrant self-renewal phenotype typically observed upon  in HSCs ,48,49. Iepletion . On the activity ,51, resuactivity . SimilarG levels . The intpatients ,54. Finapatients . However (HIF1α) .TET2 lacks the low-affinity (CXXC) DNA binding domain, which is present in TET1 and TET3 [Regulation of TET activity by controlling enzymes’ genomic distribution allows surgically modifying DNA methylation at particular genomic loci and only in specific cell types. Since and TET3 , the enzTet2 interactors/recruiters playing relevant roles in different biological settings  and Rinf (CXXC5), potentially influencing Tet2 functions in differentiation [Tet2 and Nanog distal regulatory regions of relevant pluripotency genes , positively regulating their transcription. The RNA-binding protein PSPC1 associates with Tet2 and targets the enzyme to the MERVL endogenous retroviral elements in ESCs [Tet2 contributes to their transcriptional and epitranscriptomic regulation by recruiting HDACs to chromatin and oxidizing MERVL transcripts, respectively [Additionally, ntiation  and plurntiation , respect in ESCs . Of note in ESCs ,75. Onceectively .Tet2 is also required during experimentally induced pluripotency establishment. To uncover factors associated with Tet2 in this biological setting, we analyzed DNA (hydroxy)methylation dynamics during rapid and highly efficient iPSC reprogramming systems [Tet2 is recruited by the Klf4 and Tfcp2lƒ1 transcription factors to drive active enhancer demethylation of chromatin and pluripotency-related genes to reprogram cell fate [As mentioned above,  systems ,60,76. Hell fate .Of note, a prominent study has recently identified common molecular mechanisms controlling DNA methylation during embryo development and in the leukemic transformation . Thus, hTet2 protein deficiency can lead to aberrant cell fate decisions by extensively altering the DNA methylome during hematopoiesis. Here we recapitulate the role of Tet2 recruitment to specific genomic regions in different hematopoietic developmental pathways: Tet2 role in hematopoiesis and its influence on the DNA methylome has also been widely explored in the context of physiological and pathological conditions. Several studies have described how Tet2 expression [Klf4 or Chd7, driving their active DNA demethylation and subsequent enhancer activation [TET2 [PTPN22, RUNX1, and RUNX3, among others) during hematopoietic differentiation [WT1) gene encodes a sequence-specific transcription factor found mutated in a mutually exclusive manner with TET2 in AML patients [TET2 [BTRC, DACT1, and TBL1X), preventing AML onset [Tet2-mediated epigenetic gene regulation is crucial during myeloid cell development. C/EBPα is an essential factor in the differentiation process from HSPCs to GMPs . C/EBPα pression  and, thrtivation . RUNX1, on [TET2 . Thus, intiation . Wilm’s patients . Interests [TET2  to regulML onset ,82.TET2 activity is not only relevant during early myeloid commitment but also in myeloid terminal differentiation [TET2 to promoters of key osteoclast-genes , leading to their demethylation and cell fate transition [TET2 at genomic loci exclusively demethylated in tolDCs [TET2 and initiate DNA demethylation at both EGR2 stable and transient binding sites [TET2 genomic recruitment is not always associated with positive regulation of gene expression in dendritic cells. For instance, Zhang and co-workers identified that IκBζ-dependent Tet2 targeting the Il6 locus leads to Hdac2 recruitment. Thus, finally leading to Il6 gene repression and inflammation resolution [However, ntiation . In thisansition . Tolerogn tolDCs . In adding sites . Howeversolution .EPOR, GATA1, and HBB [Tet2 recruitment at erythroid-specific genes. Of note, both factors have been recently described to interact upon Jak2-driven Tet2 phosphorylation [HSPC commitment towards erythroid lineage also correlates with 5hmC accumulation and increased expression of erythroid-specific genes such as  and HBB . 5hmC acrylation . Tet2 might contribute to the process by interacting with B-cell master regulators such as EBF1 [IgK or Aicda) [B cell differentiation is tightly regulated at the epigenetic level. B cell maturation is characterized by extensive reshaping of the cellular methylome . Tet2 mi as EBF1 , IRF4/8,r Aicda) ,92,93.Tet1 and Tet2 are upregulated in response to the sulfhydrating nuclear transcription factor Y subunit beta (NFYB) [Tet1 and Tet2 are targeted by the activated forms of Smad3 and Stat5 to the Foxp3 promoter favoring its hypomethylation and stable gene expression [5hmC is accumulated at genes encoding key regulators of T cell identity, development, and differentiation . Howevera (NFYB) . Tet1 anpression .TET2 are recruited to the specific genomic regions to repair the damaged DNA properly. TET2 depletion leads to reduced 5hmC levels at the damaged regions and impaired repair [Tet2 to protection against abnormal DNA methylation during stress have been proposed. For instance, Zhang and co-workers showed that upon OS, TET2 interacts with the thymidine glycosylase (TDG), an enzyme involved in the DNA active demethylation pathway. Then, both proteins are recruited to chromatin in a DNMT1 dependent manner, a process that is enhanced when TET2 is acetylated [Tet2 has also been implicated in genome stability. Upon exposure to oxidative stress (OS), protein complexes containing DNMTs and d repair ,96,97,98etylated . TherefoTet2 interactor in the context of DNA damage response is the SMAD nuclear interacting protein 1 (SNIP1) that regulates the expression of relevant c-MYC target genes involved in apoptosis. Therefore, reduced SNIP1 levels lead to diminished 5hmC levels and gene expression at c-MYC target genes [Another et genes .Finally, chemotherapeutic drugs have also been described to trigger hydroxymethylation changes in a mechanism mediated by the interaction between the promyelocytic leukemia protein (PML) and TET2, linking the protein to direct clinical applications .TET2 loss of function mutations are frequently found in patients suffering from myeloid malignancies such as acute myeloid leukemia, myeloproliferative neoplasms, or myelodysplastic syndromes [TET2 mutations (roughly 80%) among all blood cancer types , or myelodysplastic syndromes (MDS) ; transcription modulators ; nuclear import receptors ; and myeloid cytokines (CSDA) [As previously mentioned, es (MDS) . These cfunction ,103. Thipatients ,132  have been associated with DNA hypomethylation ,139,140.Tet2 deficient mice showed promoter hypermethylation and defective transcription factor binding at essential B-cell pathway genes. Tet2 knockout mice partially phenocopied DLBCL patients, characterized by downregulation of antigen presentation genes/interferon pathway, lymphoma-like transcriptional profiles similar to CREBBP-mutant patients, and a failure at the germinal center exit. Therefore, this data suggests that TET2 is relevant in B cell lymphoma development and how acquiring mutations in HSCs might influence B-cell maturation and cancer development [TET2 has also been studied in the context of lymphoproliferative diseases. Diffuse large B-cell lymphoma (DLBCL) patients have shown specific hypermethylation signatures on promoters of tumor suppressor genes involved in cell fate and cell cycle changes . Germinaelopment ,106.TET2 in normal hematopoiesis and blood cancer development. However, as described in this manuscript, a holistic approach is required to deconvolute the intricated regulatory networks determining TET2 function in a cell type-specific manner. Precisely, TET2 activity is controlled by: (1) TET2 transcriptional regulation ; (2) post-transcriptional regulation ; (3) post-translational regulation  enz of PTMs .Tet2 gene expression by directly recruiting Tet2 enzyme to demethylate its own enhancers during myeloid cell fate specification [Tet2 regulatory networks. Another interesting example of multilayer regulation is the miR-9/miR-34a-SIRT1-TET2 network. Sun and co-workers identified miR-9/miR-34a overexpression within a subset of TET2-WT MDS patients displaying a TET2 loss of function phenotype [TET2 activity by mediating deacetylation at key lysine residues of the enzyme [C/EBPα regulating fication  perfectlhenotype . miR-9/me enzyme . TET2 post-translational regulation, a comprehensive characterization of all potential PTMs described  to drive active DNA demethylation in a cell state-specific manner. TET2 promiscuous behavior might be encoded in its nucleotide sequence where only part of the catalytic domain (coding for the cysteine-rich domain and the first double-stranded β-helix) is structured and ordered (TET2 described interactions through a mechanism of liquid-liquid phase separation (LLPS). Of note, IDR-mediated LLPS events have been recently described for the TET2 recruiter KLF4 and its associated pluripotency transcription factor OCT4 [The rapidly increasing list of partners/interactors identified highlights the idea that tor OCT4 ,146. TET2 activity is essential to fully uncover its contribution to clonal expansion and malignant transformation.To sum up, fine dissection of the molecular mechanisms underlying


We develop this concept for Roquin and Regnase-1 function in resting and activated T cells and discuss the involvement in autoimmunity as well as how the therapeutic potential can be used in anti-tumor therapies.Post-transcriptional gene regulation by RNA-binding proteins (RBPs) is important in the prevention of inflammatory and autoimmune diseases. With respect to T cell activation and differentiation, the RBPs Roquin-1/2 and Regnase-1 play pivotal roles by inducing degradation and/or translational silencing of target mRNAs. These targets encode important proinflammatory mediators and thus Roquin and Regnase-1 functions dampen cellular programs that can lead to inflammation and autoimmune disease. Recent findings demonstrate direct physical interaction of both RBPs. Here, we propose that cooperativity of  Again, such cis-element can be composed of binding sites for one or even several RBPs to function as a regulatory unit. The site-specific recognition then induces RBP-dependent types of post-transcriptional gene regulation. Prominent cis-elements for RBPs are adenylate-uridylate rich elements (AREs) or stem-loop (SL) structures  non-coding RNAs that, together with Argonaute (Ago) proteins, form the so-called miRISC complex, which recognizes sequence-specific sites in the 3´-UTR of their target mRNAs via base-pairing  in secondary lymphoid organs. As part of the adaptive immune response, T cells reprogram their metabolism, enter and progress in the cell cycle and commit to differentiation programs that lead to specific effector or memory functions. T cell receptor (TCR) signal transduction causes epigenetic changes and induces chanisms , 2. Tranructures . Besides-pairing . Post-tr-pairing , 9. Dysr-pairing .Il2 gene contains a composite cis-element in the promoter. This cis-element requires the coinciding binding of NFAT, AP-1 and NF-κB, which are induced only during productive activation of T cells through both signals -2 requires TCR engagement and costimulation, since the  signals , 12. In  signals –16. In Ttivation . On the tivation .trans-acting factors can, by engaging in a few different combinatorial activities that have been selected in evolution, allow for a number of fine-tuned, alternative and even opposing cell fate decisions.These well-investigated examples on the regulation of transcription illustrate how a limited set of cis-element where either both RBPs bind to the RNA of the cis-element at the same or at different binding sites or by forming a complex in which just one RBP binds directly to the RNA  are often unstable, contain long 3’-UTRs with multiple binding sites for several different ng sites –28.2. The high number of approximately 1200 canonical RBPs harboring a defined RNA-binding domain (RBD) and non-canonical RBPs without defined RBDs in human or mouse primary CD4+ T cells suggests an unexplored complexity of post-transcriptional gene regulation .trans-acting factors. It responded to regulation by Roquin, Regnase-1, miRNAs and Wtap/m6A during the activation of murine T cells  In addition to the ROQ RNA-binding domain (RBD) that specifies the binding of Roquin-1 and Roquin-2 to RNA , 41–44 a2.) Roquin-1, Roquin-2 and Regnase-1 are all cleaved by the paracaspase MALT1 in response to TCR stimulation , 38.Icos, cRel, Il6, Nfkbid and also Zc3h12a, the Regnase-1 encoding mRNA (3.) Roquin-1, Roquin-2 and Regnase-1 have an overlapping set of mRNA targets including ing mRNA , 45, 47.4.) Global mapping of binding sites of overexpressed Regnase-1 crosslinked to cellular mRNAs revealed comparable sequence determinants of Roquin-recognized SLs as defined by the constitutive decay element (CDE)  , 47.5.) Roquin-1 and Regnase-1 have been found to induce post-transcriptional repression through rapid mRNA degradation as well as translational silencing , 48–50.Rc3h1 and Rc3h2 (DKO), or the Regnase-1 encoding gene Zc3h12a (KO) develop comparable phenotypes characterized by spontaneous activation of CD4+ and CD8+ T cells and accumulation of Tfh and GC B cells (6.) Mice with T cell-specific deletions of the Roquin-1/2 encoding genes,  B cells , 38, 40.Rc3h1 gene that exchanges methionine 199 to arginine (M199R) in the Roquin-1 protein develop a severe systemic lupus erythematosus-like (SLE-like) phenotype, showing deregulation of the immune system and production of anti-nuclear antibodies (ANAs), a phenotype which can be also found in the Regnase-1 KO mouse (7.) Mice harboring a single point mutation in the KO mouse , 39, 45.This striking resemblance in Roquin-1 and Regnase-1 protein functions as well as mouse model phenotypes led to the hypothesis that these two proteins may have a cooperative function. Indeed, initial data with reporter assays using overexpressed RBPs showed impaired regulation of a CDE-containing TNF 3´-UTR fragment in the absence of one or the other, supporting the idea of cooperativity .The proposed concept of cooperation , 51 has via a common SL at different times, in different subcellular locations and through different mechanisms , which was shown to be inactive in the regulation of the Tnfrsf4 mRNA, retained a residual function to cooperatively repress ICOS or Zc3h12a mRNA in reconstitution experiments  using Biacore measurements. Finally, a CDE-like SL (nt 194–212) of the Zc3h12a mRNA  MALT1 cleavage fragment when low amounts of induced and newly synthesized full-length Regnase-1 become available. This might reflect an important safe-guard mechanism which protects cells from overshooting Regnase-1 levels. Finally, upon removal of TCR signals, when Roquin and Regnase-1 re-appear in their full-length forms, they can switch off expression of cooperatively as well as Roquin and Regnase-1 independently regulated targets, efficiently stopping expression of pro-inflammatory mediators by several different modes of repression , SFB 1054-210592381 (project A03) as well as HE3359/7-1 (432656284) and HE3359/8-1 (444891219) as well as grants from the Wilhelm Sander (2018.082.2), Fritz Thyssen (Az.10.16.1.021.MN), Else Kröner-Fresenius (2015_A158) and Krebshilfe (70113538) Foundations.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.


Endocrine therapy is a standard treatment offered to patients with ERα (estrogen receptor α)-positive breast cancer. In endocrine therapy, ERα is either directly targeted by anti-estrogens or indirectly by aromatase inhibitors which cause estrogen deficiency. Resistance to these drugs (endocrine resistance) compromises the efficiency of this treatment and requires additional measures. Endocrine resistance is often caused by deregulation of the PI3K/AKT/mTOR pathway and/or cyclin-dependent kinase 4 and 6 activities allowing inhibitors of these factors to be used clinically to counteract endocrine resistance. The nuclear mechanisms involved in endocrine resistance are beginning to emerge. Exploring these mechanisms may reveal additional druggable targets, which could help to further improve patients’ outcome in an endocrine resistance setting. This review intends to summarize our current knowledge on the nuclear mechanisms linked to endocrine resistance. Breast cancer (BC), a systemic disease characterized by early tumor cell dissemination , is the Routine treatment options for BC patients include ERα- and Her2-targeting therapies, chemotherapy, surgery and radiation. Besides ERα and Her2 expression, the luminal subtype, tumor grading and lymph node involvement play a role in therapy decision . Endocriesr1 (Endocrine resistance (ENDO-R), the resistance to ERα-targeting therapy, is a major obstacle in treatment of ERα-positive BCs. In first-line treatment, ENDO-R is observed in approximately half of all ERα-positive BCs . Many faesr1 –19, thisThere are two estrogen receptors, ERα and ERβ . While Eesr1) and by proteasome-dependent degradation of the ERα protein  gene coding for the PI3K catalytic component p110α , Her2, Her3, Her4, fibroblast growth factor receptor (FGFR), insulin-like growth factor receptor (IGF1R) and insulin receptor (IR) , in combination with endocrine therapy are currently standard of care for advanced ERα-positive breast cancer , 65–68. Deciphering the changes happening in the nucleus upon acquisition of ENDO-R may result in the identification of additional druggable factors in ENDO-R.Chromatin Accessibility). Both types of TFs are involved in ENDO-R (Sequence-specific transcription factors (TFs) are key drivers of gene expression and can have activating or repressive functions. Activating TFs induce gene transcription by binding to promoters and/or enhancers followed by recruitments of co-activators and RNA polymerase . Two majATF2 and c-Jun are members of the AP-1 family of transcription factors and often form heterodimers , 78. ImpA screen in a chemical library resulted in the discovery of two ATF-2 inhibitors, celastrol (CSL) and acetyl isogambogic acid (AIGA), which both proved to be potent inhibitors of melanoma growth . CSL, knElf5, also known as epithelium-specific Ets transcription factor 2 (ESE2), a member of the E26-transformation-specific/E-twenty-six-specific sequence (ETS) domain family of transcription factors , plays aERRα is an orphan nuclear receptor that shows a high homology to ERα in the DNA binding domain, potentially allowing ERRα to activate ERα-target genes in the absence of estrogen . Higher in vitro and in vivo , the human homologue of XAG-2, a elopment . AGR2 iselopment . In murielopment . AGR2 iselopment  and the elopment , being celopment .As shown with MCF-7 cells, AGR2 is important for ERα-driven proliferation , 116–120Higher AGR2 expression is associated with unfavorable prognosis in BC . This hoAntibodies against AGR2 and LYPD3 have been found to be effective to suppress growth of TAM-R breast cancer cells in mice . AdditioThe FoxM1 gene is transcriptionally regulated by ERα and is important for ERα-driven cellular growth . AccordiFoxM1 may be suitable as a predictive marker in ENDO-R. Overexpression of FoxM1 in ERα-positive breast cancer was found to correlate with worse prognosis of TAM-treated patients , 126. InFoxM1 might be targeted through 14-3-3ζ, whose activity can be inhibited by small molecules, such as FOBISIN101, or by the peptide inhibitor R18 . R18 wasCancer Stem Cells).Hypoxia stabilizes HIF-1α and HIF-2α proteins allowing them to initiate transcription of numerous genes engaged to ensure survival under hypoxic conditions . In cancOverexpression of HIF-1α or HIF-2α was found to desensitize MCF-7 cells to FULV , 136. LiOne target of HIF-2α is EGFR, which has been linked to anti-estrogen resistance. EGFR can also feedback on HIF-2α . Furtherin vitro and in vivo  or carcinoma-associated fibroblasts (CAFs) the atypical IκB protein B-cell lymphoma-3 (Bcl-3) plays a role, whose expression is associated with poorer survival of TAM-treated patients . Bcl-3 cA number of drugs interfering with the NFкB pathway have been developed . some ofesr1, the gene coding for ERα, another the gene encoding the NFкB transcription factor p65/RelA (Unfolded protein response (UPR) is activated in the event of endoplasmic reticulum stress . UPR is p65/RelA . Overexpp65/RelA , while ip65/RelA , likely p65/RelA . Higher p65/RelA .The importance of UPR for drug resistance has planted the idea of inducing an overload of ER stress . This coThe transcription factor YB-1, a so-called cold-shock protein, is involved in cellular stress responses . By bindIts role in ENDO-R is further supported by the finding that ectopically expressed YB-1 desensitizes MCF-7 and T47D cells to FULV and TAM , 180. Lain vitro and in vivo.Interference with YB-1 activity is possible by the novel multikinase inhibitor TAS0612, which targets AKT, p70S6K, and p90RSK and thereby prevents YB-1 phosphorylation at Ser102 and its subsequent transport into the nucleus . TAS0612Chromatin accessibility is defined by the ability of DNA-binding factors to access chromatin DNA, which is highly compacted by its interactions with histones and other chromatin-binding factors . Chromatde novo DNA methylation in tumorigenesis prevent the activation of key genes involved in terminal differentiation and thereby in inhibition of proliferation  may be of particular importance in FULV-R -inducing-TFs .The Notch signaling pathway, important for the maintenance of CSC activity in BC , has beenotch4 gene involves the transcriptional repressor death domain associated factor 6 (DAXX), a protein stabilized by ERα. In turn, DAXX recruits DNMT1 to the notch4 promoter leading to DNA methylation. Besides Notch4, DAXX also down-regulates other stemness-relevant genes, including ALDH1A1, thereby causing the tumor-initiating capacity of BC cells to decline. Importantly, higher expression of DAXX correlates with more favorable outcome of patients who received endocrine treatment .The Wnt pathway is an important pathway in mammopoiesis, involved in mammary stem cell regulation and cell fate decisions . Its derIn FULV-R and TAM-R MCF-7 sublines, expression of Wnt pathway components, including β-catenin, are increased . Also, o+/CD24- CSCs (EMT is an essential process in embryonal development and wound healing allowing stationary cells to switch to a migrating phenotype . This is24- CSCs . EMT may24- CSCs . Quasi-mesr1 promoter and to inhibit ERα expression, leading to estrogen-independent proliferation and FULV and TAM resistance (esr1 transcription coincided with DNA methylation, caused by the Twist-recruited DNMT3B. Like Twist, ZEB1 induces esr1 promoter hypermethylation and ENDO-R (Overexpressed in MCF-7 and T47D cells, Twist was shown to bind to the sistance . Twist-id ENDO-R , while Zd ENDO-R . In FULVd ENDO-R .Clinically, higher expression of Twist, Snail or Slug was found to be associated with a higher probability to relapse in ERα-positive BCs . Twist aCurrently, the PI3K/AKT/mTOR pathway and CDK4/6 are the prime targets to manage endocrine resistance. However, inhibitors against these targets may fail. For instance, ENDO-R resulting from Her2 mutations or FGFR1 amplification are also resistant to the CDK4/6 inhibitor palbociclib , 240. HeFurthermore, by exploring the nuclear mechanisms that allow escape from endocrine treatment new druggable targets may come to light. However, the diversity of nuclear mechanisms leading to ENDO-R requires additional diagnostics to clarify which nuclear changes are responsible for the observed resistance. One such recently identified promising target is the histone PTM reader BRD4 , whose aTranscription factors are more difficult to target, as they usually lack enzymatic activity. However, drugs can interfere with these factors indirectly, for instance, by blocking enzymes responsible for their activation or by inhibiting their interactions with essential co-factors. Transcription factors that are druggable through such an approach include YB-1, NFкB, Notch and ERRα. YB-1 activity can be suppressed by blocking the kinases that catalyze an essential activating phosphorylation event . NFкB caTranscriptional activities could also be controlled by interfering with certain miRNAs. For instance, by blocking miR221/222 β-catenin-dependent transcription FULV resistance can be suppressed .Thus, understanding the nuclear mechanisms involved in ENDO-R may help to dissect those patients who benefit most from treatment with PI3K/AKT/mTOR pathway and CDK4/6 inhibitors and, additionally, may allow the identification of new druggable targets.The author confirms being the sole contributor of this work and has approved it for publication.The author declares 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.


Transcription factors (TFs) bind DNA in a sequence-specific manner and are generally cell type-specific factors and/or developmental master regulators. In contrast, general TFs (GTFs) are part of very large protein complexes and serve for RNA polymerases’ recruitment to promoter sequences, generally in a cell type-independent manner. Whereas, several TFs have been proven to serve as anchors for the 3D genome organization, the role of GTFs in genome architecture have not been carefully explored. Here, we used ChIP-seq and Hi-C data to depict the role of TFIIIC, one of the RNA polymerase III GTFs, in 3D genome organization. We find that TFIIIC genome occupancy mainly occurs at specific regions, which largely correspond to Alu elements; other characteristic classes of repetitive elements (REs) such as MIR, FLAM-C and ALR/alpha are also found depending on the cell’s developmental origin. The analysis also shows that TFIIIC-enriched regions are involved in cell type-specific DNA looping, which does not depend on colocalization with the master architectural protein CTCF. This work extends previous knowledge on the role of TFIIIC as a bona fide genome organizer whose action participates in cell type-dependent 3D genome looping via binding to REs. Three-dimensional folding of the mammalian genome acts as a decisive regulatory layer for determining important physiological processes such as cell fate, development, differentiation, and ultimately even pathological conditions such as tumorigenesis, by implementing cell-type specific gene expression programs . SeveralDEEs are loaded with a plethora of CTFs , which uA generally accepted model for the communication between DEEs and CPEs proposes that CTFs and GTFs are linked by a bridge such as the Mediator coactivator complex , pointinTo explore TFIIIC chromatin behavior in several human cell lines, we used TFIIIC genome-wide ChIP-seq data from fetal lung fibroblasts (IMR90), normal mammary gland epithelial cells (MCF10A), infiltrating ductal carcinoma breast cancer cells (T47D) , and humRNA7SL1  and one unique tRNA gene (tRNA-Glu-GAG)  B. Of theGlu-GAG) C. A largGlu-GAG) C. HoweveGlu-GAG) A. In ordGlu-GAG) D. As in Glu-GAG) D.p-value < 0.001) enrichment for genes associated with cadherin adhesion  are cis genetic fingerprints of regulatory DNA elements shown to possess cell type- and state-specificity in humans . In addiAs cell type-specific TFIIIC-bound regions are both AE and non-AE, we wondered which group contributed most to the overlap with DHSs. The intersected of the different datasets showed that the AE fraction provided the greatest contribution to the DHS overlap for IM3C and ES3C, with 809 DHS-positive regions out of 1037 AE (78%) and 985 DHS-positive regions out of 1162 AE (85%), respectively . The oveWe have previously reported that TFIIIC controls gene expression via mechanisms involving gene looping and histone acetylation in response to serum starvation in T47D cells . As manyThe most studied genome organizer is known as CCCTC-binding factor (CTCF) ,40. TherTo further prove that CTCF is not involved in looping generated at cell type-specific TFIIIC-bound regions, we calculated the average profile of CTCF at MC3C, IM3C, ES3C and TD3C. We found that CTCF was indeed not significantly enriched at any of the cell type-specific TFIIIC-bound regions E. A slig3D genome organization plays a fundamental role in regulating gene transcription ,45. HoweTFIIIC recognizes two DNA sequences known as A- and B-box, which are the prototypical internal promoter of Pol III transcriptional units , and areFinally, a general conclusion of our findings is that cis-regulatory elements within the mammalian genome are not such well-defined entities as previously thought, and many more TFs, which have been mis-categorized as housekeeping GTFs, such as TFIIIC, might instead play more cell-type specific functions in the DEEs and promoters’ interaction.Human H9 (WiCell) ES cells were grown feeder-free on the matrigel  coated 6-well plate in the maintenance media . They were passaged with enzyme-free method  once they become nearly confluent. On the day of passaging, the media is supplemented with 10 µM Y27632 .Cross-linked chromatin free of RNA from hESC H9 was prepared in the lab of Atsushi Nakano at the University of California Los Angeles (UCLA), Los Angeles (USA), and immunoprecipitation was carried out with the TFIIIC antibody  and perfSingle-end sequencing libraries were constructed from 1 ng of immunoprecipitated and input DNA using the Ovation Ultralow DR Multiplex System 1–8 and 9–16 kit (NuGen). To minimize false positives calling, several input libraries were sequenced to reach saturation with a coverage of 4 reads/bp of the human genome for each condition.ChIP-seq data for GTF3C in IMR90, MCF-10A and T47D was taken from GEO: GSE120162. ChIP-seq for GTF3C in hESCs was produced in this study (deposited at GEO repository with ID number: GSE195499). ChIP-seq for CTCF in T47D was taken from GEO: GSE120162; in MCF-10A from GEO: GSM2599084; in IMR90 from GEO: GSM935404; in hESCs from GEO: GSM803419. ChIP-seq for Pol2 in T47D was taken from GEO: GSM1503827; in MCF-10A from GEO: GSM2467771; in IMR90 from GEO: GSM935513; in hESCs from GEO: GSM3395077.Analysis of ChIP-seq data was carried out as previously described  with minhttps://www.rstudio.com, accessed on 22 January 2022).Bed intersection was carried out using bedtools  “intersehttp://chip-enrich.med.umich.edu, accessed on 22 January 2022) [ChIP-seq Enrich (ry 2022)  was usedhttps://hichip.readthedocs.io/en/latest/index.html, accessed on 26 January 2022). Hi-C matrixes were generated from HiChIP-seq data using HiC-pro pipeline [https://bioconductor.org/packages/release/bioc/html/Sushi.html, accessed on 22 January 2022) and aligned with ChIP-seq data and Hi-C matrices. Finally, hicAggregateContacts plots were generated using HiCExplorer tools [HiChIP-seq analysis was performed using dovetail HiChIP pipeline using manufacturer’s instructions (pipeline . Finallypipeline . Loops rer tools .This work extends previous knowledge about the role of TFIIIC as a bona fide genome organizer whose action participates in cell type-dependent 3D genome looping via binding to REs. This work also emphasizes the role of diverse REs in regulating chromatin folding defying an architectural role for SINEs, FLAM-C, MIR, ALR/alpha in human cells via binding of TFIIIC. Our findings expose novel roles for this general transcription factor that could go far beyond its known function in Pol III transcription. Therefore, our data points to TFIIIC as a potential new “integrator” capable of reading different and dispersed REs within the genome and rewiring them into a cell type-specific network of CTCF-devoted loops in human cells.


Circadian rhythms orchestrate organismal physiology and behavior in order to anticipate daily changes in the environment. Virtually all cells have an internal rhythm that is synchronized every day by Zeitgebers . The synchrony between clocks within the animal enables the fitness and the health of organisms. Conversely, disruption of rhythms is linked to a variety of disorders: aging, cancer, metabolic diseases, and psychological disorders among others. At the cellular level, mammalian circadian rhythms are built on several layers of complexity. The transcriptional-translational feedback loop (TTFL) was the first to be described in the 90s. Thereafter oscillations in epigenetic marks highlighted the role of chromatin state in organizing the TTFL. More recently, studies on the 3D organization of the genome suggest that genome topology could be yet another layer of control on cellular circadian rhythms. The dynamic nature of genome topology over a solar day implies that the 3D mammalian genome has to be considered in the fourth dimension-in time. Whether oscillations in genome topology are a consequence of 24 h gene-expression or a driver of transcriptional cycles remains an open question. All said and done, circadian clock-gated phenomena such as gene expression, DNA damage response, cell metabolism and animal behavior—go hand in hand with 24 h rhythms in genome topology. Our environment displays multiple cycles with different periodicities, from ultradian , circadian , and infradian . Oscillations in light/dark cycles, availability of food and temperature rhythms are some of the most common circadian Zeitgebers (time-giver) encountered by organisms. Hence, virtually all species have evolved a circadian clock to adapt and anticipate such changes in environmental conditions. The presence of a circadian rhythm is defined on the basis of three criteria i) free-running period of ∼24-h, ii) ability to entrain to environmental cues, and iii) rhythms that are temperature compensated.Clocks in different organisms seem to be the result of convergent evolution as the molecular core-clock components have diverged across the kingdoms of life. Circadian rhythms appeared in an ancestor of present-day cyanobacteria where the oscillator is built atop a post-translational phosphorylation-dephosphorylation cycle of the Kai A-B-C complex . It appeIn Situ Hybridization (FISH)  . This ten (FISH) . The firn (FISH) . Then win (FISH) , multipln (FISH) , and finn (FISH) . These tn (FISH) . The pren (FISH) .This leap in technology gives a good overview of the genome organization in the nucleus and allowed one to ask if topological features are static or dynamic over time. Recent studies have focused on the genome topology reorganization under particular biological conditions such as during development, in aging, after an external cell stimulus, or in disease. A growing body of evidence now suggests that topological genome organization is rhythmic over 24 h-i.e. it cycles back to its original state at the start of every day. Thus, not only could this phenomenon be central in establishing circadian cycles in gene expression but also could be used in the daily control of DNA damage protection and/or repair pathways or regulation of aging over animal lifetimes.In this review, we will focus on the core-mammalian clock mechanisms, followed by a discussion on the current evidence on how genome topology could be integrated into the mechanism of the core circadian clock. We will end by discussing the consequences of cycling genome topology on cellular functions.The mammalian clock is built on an evolutionarily conserved negative TTFL  which isThe cyclic activation-repression of gene expression by the core clock transcription factor machinery relies on the secondary structure of the genome-the chromatin landscape. Genome compaction is achieved by the packaging of DNA into a repeated array of nucleosomes composed of histone octamers containing two molecules each of histones H2A, H2B, H3, and H4  or their variants  . The pacDuring the activation phase, the BMAL1-CLOCK complex has been shown to recruit lysine-specific histone demethylase JARID1A and LSD1 that could remove the repressive H3 lys9 di- and tri-methyl (H3K9me2/3) marks . The disTo prepare the repressive phase, the BMAL1-CLOCK complexes mark nucleosomes with ubiquitin moieties at their target sites with the aid of DDB1-CUL4 complexes. The resulting mono-ubiquitinated H2B nucleosomes participates in PER complex recruitment . The repIn addition to post-translational modifications, canonical histones can also be exchanged for histone variants. The variant histones have specific characteristics compared to canonical counterparts. In addition to not having the same residues for post-translational modification they also have varying affinity of interactions with other histones, DNA and act as a scaffold for chromatin interactor recruitment. The histone variant H2A.Z has been proposed to participate in chromatin opening due to a higher density of these nucleosomes around BMAL1 binding site following TF binding  but funcFinally, remodeling of nucleosome array can also participate in the activation and repression of circadian gene expression. At the beginning of the activation phase, the BMAL1-CLOCK complex, which is thought to act as a pioneer factor, recognizes E-box sequences engaged within nucleosomes in facultative heterochromatin and favors nucleosome eviction creating nucleosome depleted regions . AssociaExperiments to determine the full repertoire of chromatin modifiers and remodelers that are at the core of the circadian oscillator are still ongoing. The mechanism by which cells coordinate gene-specific remodelers at the genome-scale is likely linked to the higher layer of gene expression control - at the level of 3D organization of the genome.Starting from a linear fiber of DNA to the highly condensed mitotic chromosome, there exists several intermediate levels of chromatin organization. Chromatin forms loops which can be grouped into TADs and sub-TADs are split between compartment A  and compartment B  . TADs caDbp locus as a viewpoint, interactions with 200 genomic interchromosomal loci were observed in all, of which 29 were oscillating. Knocking out Bmal1 in these cells specifically disrupted the oscillating interactions revealing the need for the core clock in the dynamic Dbp gene interactome. Investigations at the Nr1d1 locus (coding REV-ERBα) revealed stable interactions during the circadian cycle that are important for the cyclic activation of transcription . The ficription . Cohesincription .Furthermore, the Lazar group deployed the Hi-C method to define interactions between two genomic loci  at two dBmal1 Per1 and Per2 were shown to be involved in oscillating interactions with surrounding enhancers. The time of the highest interactions between promoter and enhancers precedes pre-mRNA synthesis. To determine if oscillating genomic contacts are specific to the core clock or general among oscillating genes, several output gene loci were also considered . Most of these loci showed stable interactions with enhancers over time and importantly these were observed also in arrhythmic Bmal1−/− animals. Output genes seem to arrange in a chromatin hub of temporally co-transcribed genes. An independent study from the Fraser group arrived at a similar conclusion but at a genome-wide scale . LADs are typically heterochromatic in nature. In 2015, Zhao et al. demonstrated that PARP1 targets CTCF-bound chromatin to LADs to inhibit circadian gene expression . The expr tissue .Additional work on the nuclear location of core-clock and output gene loci will be required to understand the roles of the interactions. Are genomic interactions by themselves the crucial topological features of the core clock and output gene expression? Or do genomic interactions participate in relocation of the loci in 3D space required for gene repression? It is tempting to speculate that because core clock genes form transient loops during the day, the enhancer-promoter interactions are directly involved in the regulation. Could the hub could move from euchromatin territories to heterochromatin territories like the lamina or around the nucleolus? The requirement of chromatin topology modifications to build the circadian core clock is fertile for the next phase of investigations.The circadian clock integrates several layers of regulation from the activator-repressor complexes to genome topological oscillations via chromatin state modifications. Such a robustly controlled self-sustained mechanism not only manages the expression of output genes but also could use these dynamics for other functions within cells. One such phenomenon in differentiated cells is the DNA damage and repair response.Genomic integrity is at constant risk of damage from solar ultraviolet radiation, from metabolism-derived reactive species and xenobiotics. The risk to the genome from such phenomena are spread out over 24 h and is linked to light exposure that affects exposed organs such as the skin, while activity and feeding cycles typically impact visceral tissues. A description of DNA damage pathways and damage response and repair (DDRR) is beyond the topic of this review, and we invite readers to other excellent reviews on this topic . DDRR paIt has notably been shown that condensed heterochromatin is more resistant to DNA damage and protected from double-strand breaks (DSB) while more open chromatin is more sensitive to such damage . One of Interestingly, in mice, a non-negligible part of chromatin tends to be more open during the day and more condensed during the night  and repressors  that build the loop. The discovery of chromatin organization dynamics followed after. The clock complexes recruit multiple chromatin modifiers and so epigenetic marks oscillate over 24 h. Finally, it is only recently that genome topology dynamics have been implicated in the core clock mechanism with specific features that distinguish it from the outputs of the clock. The core clock loci form few and dynamic contacts whereas output loci make numerous but stable interactions. Nevertheless, work is still required to understand the requirement of genome topology in the circadian clock and if the folding of the chromatin is sufficient or if the spatial displacement of circadian regions in nuclear space is also a prerequisite. Whether pathways that are identified in the liver or cell lines apply to all tissues/cells where peripheral clocks function also remains open to study.Bmal1 knockouts) age extremely rapidly (How does the aging clock alter chromatin and genome topology? During aging, rhythms tend to weaken with damped amplitudes and shorter periods  and at i rapidly . Cells f rapidly . Neverth rapidly . Are cor rapidly , a focus rapidly .


Pichia pastoris is not only used as an excellent host for heterologous protein expression but also exhibits good potential for efficient biosynthesis of small-molecule compounds. However, basic research on P. pastoris lags far behind Saccharomyces cerevisiae, resulting in a lack of available biological elements. Especially, fewer strong endogenous promoter elements available for foreign protein expression or construction of biosynthetic pathways were carefully evaluated in P. pastoris. Thus, it will be necessary to identify more available endogenous promoters from P. pastoris.Due to its ability to perform fast and high-density fermentation, 0019, P0107, P0230, P0392, or P0785 was basically unchanged during the logarithmic phase and stationary phase of growth. And the transcriptional level contributed by P0208 or P0627 exhibited a growth-dependent characteristic (a lower expression level during the logarithmic phase and a higher expression level during the stationary phase). After 60 h growth, the β-galactosidase activity contributed by P0208, P0627, P0019, P0407, P0392, P0230, P0785, or P0107 was relatively lower than PGAP but higher than PACT1. To evaluate the availability of these promoters, several of them were randomly applied to a heterogenous β-carotene biosynthetic pathway in P. pastoris, and the highest yield of β-carotene from these mutants was up to 1.07 mg/g. In addition, simultaneously using the same promoter multiple times could result in a notable competitive effect, which might significantly lower the transcriptional expression level of the target gene.Based on RNA-seq and LacZ reporter system, eight strong endogenous promoters contributing to higher transcriptional expression levels and β-galactosidase activities in three frequently-used media were screened out. Among them, the transcriptional expression level contributed by PP. pastoris. And the competitive effect observed here suggests that a careful pre-evaluation is needed when simultaneously and multiply using the same promoter in one yeast strain. This work also provides an effective strategy to identify more novel biological elements for engineering applications in P. pastoris.The novel strong endogenous promoter identified in this study adds to the number of promoter elements available in The online version contains supplementary material available at 10.1186/s12934-021-01648-6. Pichia pastoris  is not only widely used as an excellent host for heterologous protein expression [P. pastoris, compared with Saccharomyces cerevisiae, fewer strong endogenous promoter elements available for foreign protein expression or construction of biosynthetic pathways were carefully investigated in P. pastoris [As one of the typical methylotrophic yeasts, pression  but alsopression , carotenpression , 4, xantpression , ricinolpression , hyaluropression  and dammpression . Althougpastoris .P. pastoris. Several promoters from the methanol utilization (MUT) pathway were identified as the methanol-dependent strong promoters [AOX1  [FLD1  [DAS (the promoter of dihydroxyacetone synthase) [PEX8  [AOX2  [Using methanol as a carbon source is one of the most favorable properties of romoters , such asidase 1) , PFLD1 (enase 1) , and PDAynthase) . On the protein)  or PAOX2idase 2)  was clasidase 2) .GAP is the most frequently used constitutive promoter that drives the expression of glyceraldehyde-3-phosphate dehydrogenase at a high level in the medium with glucose or glycerol but at a moderate level in the medium with methanol as a carbon source. Although in many cases, PAOX1 works more efficiently than PGAP for heterologous protein production [GAP in the medium containing glucose was even higher than PAOX1 in the medium containing methanol [GAP regulated mammalian peptide transporters rPEPT2 by Doring F et al. [GAP was generated, and it spanned an activity range between 0.6% and 19.6-fold of the wild-type PGAP activity [TEF1, which controls the expression of translation elongation factor 1 alpha [GAP in batch cultures or fed-batch cultures (glucose or glycerol was used as carbon source). PGCW14 is a strong constitutive promoter discovered recently [TEF1 or PGAP under different carbon sources  when an enhanced green fluorescent protein (EGFP) was used as the reporter. Moreover, some other constitutive promoters, PENO1, PGPM1, PHSP82, PKAR2, PPGK1, PSSA4, and PTPI1 [GAP.Poduction , 18. Sommethanol . SimilarF et al. . A promoactivity . As anot 1 alpha , was alsrecently . It exhind PTPI1 , were alAlthough most studies have focused on strong promoters, it is worth noting that strong promoters are not suitable in some applications. For example, promoters with moderate expression levels are sometimes more desirable for transporter or chaperone expression. Thus, the studies on various promoters with different transcriptional activities should also be emphasized. In this study, based on the RNA-seq data and LacZ reporter system, eight novel endogenous promoters that exhibited strong transcriptional activities in three frequently-used media were screened out. A heterogenous β-carotene biosynthetic pathway was chosen to further evaluate the availability of these promoters. It was also found that simultaneously using the same promoter multiple times could result in a notable competitive effect, which might significantly lower the transcriptional expression level of the target gene.P. pastoris, the assessment of cell growth status was firstly carried out. As shown in Fig. In order to screen out strong promoter candidates from ACT1 (PAS_chr3_1169), which is usually used as an endogenous reference gene (housekeeping gene) in yeast [ACT1 could be considered as a strong promoter. To explore the potential strong promoters in all the above media, the genes with FPKM values five times higher than that of ACT1 were selected as the candidate genes. Finally, the promoters of PAS_chr1-4_0586 (GCW14), PAS_chr4_0627, PAS_chr2-2_0019, PAS_chr2-1_0437 (GAP), PAS_chr1-1_0407, PAS_chr2-2_0392, PAS_chr3_0230, PAS_chr2-2_0208, PAS_chr4_0785, and PAS_chr1-1_0107 were screened out . As shown in Fig. As shown in Fig. in yeast , 26, wasXXX-lacZ-TADH1 (PXXX indicates the name of candidate promoter) were measured after 60 h growth. The transcriptional levels of lacZ in all transformants were also tested by RT-qPCR. All experimental data was obtained from three independently screened colonies. As shown in Fig. ACT1 when the transformant was cultured in three mentioned broths, except P0107 which exhibited a lower β-galactosidase activity compared to PACT1 in YPM broth. In YPD or YPG broth, the activity of PGAP or PGCW14 was significantly higher than those of the rest promoters. In YPM broth, PGCW14 contributed the highest β-galactosidase activity among the all-selected promoters, and PGAP, P0208, P0019, P0392, P0230, or P0627 contributed a similar β-galactosidase activity. In summary, among the ten selected promoters, the previously reported PGAP and PGCW14 showed strong transcriptional activities. The transcriptional activity of P0019, P0107, P0208, P0230, P0392, P0407, P0627, or P0785 was between that of strong promoters PGAP and PACT1. It indicates that these promoter candidates could be recognized as strong endogenous promoters in P. pastoris.In order to compare the transcriptional activities of above-selected promoters, the β-galactosidase activities of transformants harboring vector pZeocin/P0407 and P0230. For example, the transcriptional level of P0407-lacZ was much higher than that of PGAP-lacZ when the relative transformants were cultured in YPG or YPM broth. However, the β-galactosidase activities controlled by P0407 were less than half of that by PGAP. So was P0230-lacZ. We evaluated the copy number of lacZ controlled by each promoter, the results proved all clones harboring only one lacZ, eliminating the influence of transcriptional level by copy number (data not shown). It indicated that the transcriptional and protein level were not coordinated in some genes. The reason was not clear.Although the transcriptional activities of most selected promoters corresponded with the β-galactosidase activities at 60 h, there were still few exceptions, such as PGAP being identified as a recognized constitutive promoter, it was chosen as a control in this study. As shown in Table 0407, P0627, and P0208, could be defined as constitutive promoters due to their relatively stable β-galactosidase activities at 16 and 60 h. The relative β-galactosidase activity driven by P0208, P0407, or P0627 at 16 h was at least 16.5, 2.5 or 7.5 times lower than that at 60 h in three media, reminding us that these promoters might be associated with cell growth.To evaluate whether the above promoters are constitutive, the β-galactosidase activities of different transformants at 16 h  and 60 h (stationary phase) were measured. In view of P0407, 0208, and 0627 genes in the GS115 WT strain were detected at the different growth stages. As shown in Additional file 0407, 0627 and 0208 genes increased along with the growth time of GS115 WT strain in YPD and YPG media, indicating P0407, P0208 and P0627 could be considered as growth-dependent promoters. In YPM broth, they behaved not growth-dependent at 60 h. We suspect that it may be related to the depletion of carbon source (methanol). In our study, YPD (2%), YPG (2%) or YPM (1%) has 32.58, 33.36 and 12.26 mmol carbon atoms, respectively. At 60 h, the methanol had been depleted which may affect the cell metabolism, so the relative transcriptional level of 0627, 0407 and 0208 at 60 h was lower than that at 36 h, showing non-growth-dependent characteristic. From 16 to 36 h, the relative transcriptional level was increased. Throughout the performance of 0208 and 0627 in three media for three time points, it demonstrates that they are growth-dependent. In addition, the transcriptional level of 0407 was higher than that of ACT1 at 16 h in YPD, YPG and YPM  with different transcriptional activities were randomly selected and applied to the β-carotene biosynthetic pathway to further evaluate the availability of promoters. As shown in Fig. XXX-crtE-PYYY-tHMG1 and pG418/PXXX-crtYB-PYYY-crtI, PXXX and PYYY indicate the name of candidate promoters) containing the above four genes were constructed (Step 2) and transformed into the GS115 WT strain in turn (Step 3). The promoter activities were qualitatively observed by the color of the transformants on plates (Step 4) and then quantitively determined by liquid fermentation (Step 5).According to the previous report, the optimized β-carotene biosynthetic pathway always includes four genes, tYB/crtI . In thisFinally, fourteen transformants with significant color differences were screened out for further liquid fermentation Fig. A. The recrtE, tHMG1, crtYB, and crtI) in each strain were verified by PCR and the primers were shown in Additional file crtYB and crtI had a stronger promoter (similar to PGAP), the β-carotene production of engineered strain was generally higher, and when crtYB had a relatively weaker promoter compared to PGAP, the β-carotene production of engineered strain was decreased. This result was consistent with the previous report that CrtYBp was the rate-limiting enzyme in β-carotene production [crtE. The promoter of crtE was PGAP in the Caro-13 strain and P0230 in the Caro-14 strain. Although the transcriptional activity of P0230 was lower than that of PGAP at 60 h, the β-carotene production of the Caro-14 strain was much higher than that of the Caro-13 strain. This result suggested that not all genes should have stronger transcriptional levels to achieve higher β-carotene production. Balancing the expression level of each gene in the biosynthetic pathway could be a better choice.The promoters of four genes  and two exogenous genes (crtE and crtI) were all driven by PGAP in the Caro-9 strain, the transcriptional levels of both exogenous genes were much lower than that of GAP, presenting a significant “competitive effect”. In addition, the transcriptional levels of crtE and crtI were also different. So were GCW14, crtYB, and tHMG1controlled by PGCW14. A similar phenomenon was also observed in the Caro-13 strain  which were not affected by carbon sources were screened out. Except for PGCW14 and PGAP, eight of them were first reported here. Five  were identified as strong constitutive promoters, and two (P0627 and P0208) as growth-dependent strong promoters. The growth-dependent transcriptional behavior of P0627 or P0208 makes it a good selection for the expression of toxic foreign proteins in P. pastoris. P0407 showed a similar trend as P0627 and P0208 in β-galactosidase activity. However, the transcriptional level of P0407-lacZ was higher than that of ACT1 at 16 h, indicating this promoter was not a strictly growth-dependent promoter. In addition, in our study, the activity of PGCW14 was lower than that of PGAP in YPD broth and similar to that of PGAP in YPG broth when measured with the β-galactosidase activity assay. These results were not consistent with the previous report that PGCW14 activity was much higher than PGAP by EGFP reporter in YPD or YPD broth [In this study, by analyzing the RNA-seq data of GS115 WT strain in the stationary phase under different media , ten strong endogenous promoters . The Caro-14 strain contributed the highest yield of β-carotene (1.07 mg/g DCW). It is higher than the yield in S. cerevisiae containing integrated carotenogenic gene cassettes crtYB/I/E/tHMG1 (501 µg/g DCW) [P. pastoris might have the advantages to be engineered as a carotene producer.In this study, a heterogenous biosynthetic pathway of β-carotene containing four genes was used to further evaluate the properties of six strong endogenous promoters (Pg/g DCW) , suggestIn addition, the competitive effect was first evaluated in this study. It was found that using the same promoter multiple times in one strain could lead to a significant decrease of the expression level of the target gene, reminding a careful evaluation is required before using a promoter in this way.S. cerevisiae [GAP in P. pastoris, its’ sequence length is 477 bp. It should be noted that the intergenic region between GAP and its neighbor gene was 1576 bp. On the contrary, the sequence length of another well-known strong inducible promoter, PAOX1, is 939 bp in the commercial vector pPICZαC, mainly covering the intergenic region between AOX1 and its neighbor gene (998 bp). The study of PAOX1 showed that the region − 1055 to − 809 and − 653 to − 514 significantly affects the promoter activity [AOX1, thereby affecting their normal functions. The corresponding intergenic regions of six selected promoters are over 500 bp except P0208, reminding the possibility that several key regulatory elements might not be included in the 500 bp sequence  containing 100 µg/mL ampicillin, zeocin, or 50 µg/mL kanamycin sulfate, and incubated at 37 °C. The yeast mutants were derived from the P. pastoris GS115 wild-type strain. All yeast strains, unless otherwise specified, were grown in YPD medium  at 30 °C.All strains used in this study are listed in Additional file 600 of 0.1 in 250 mL shaking flasks containing 50 mL YPD (2% glucose), YPG (2% glycerol), or YPM (1% methanol) for 72 h. The optical absorbance (OD600) of the culture was detected by an Evolution™ 220 spectrophotometer , and each medium had three replicates.The GS115 WT strain was pre-cultured in 5 mL YPD broth at 30 °C, 220 rpm for 24 h and followed by inoculating to an initial OD600 of 16) were harvested by centrifugation  at 36 h when they entered the stationary phase. Total RNA was isolated using a Yeast RNA kit  through the operation manual. The residuary genomic DNA was eliminated using a DNase I treatment . The qualified RNA samples determined with Bioanalyzer 2100  were used for library construction with the sequencing platform BGISEQ-500 . Compared to the genome, the average comparison rate of the sample is 97.13%, and the average comparison rate of the compared gene set is 82.58%. All the generated raw sequencing reads were filtered to remove reads with adaptors, reads with more than 10% unknown bases, and low-quality reads. Clean reads were then obtained and stored in FASTQ format. The clean reads were mapped to the genome of GS115 by HISAT [ACT1 was used as the relative fold change for RNA-seq data treatment.Three independent clones of the GS115 WT strain were incubated in 5 mL YPD broth for 24 h. The pre-cultured pellet was washed with presterilized water and transferred into YPD, YPG, or YPM broth for incubating. The yeast cells . RT-qPCR was carried out on a Mastercycler® ep gradient S instrument  with 2 × ChemQ SYBR qPCR Mester Mix . The analysis .E. coli BL21 (DE3) was used as a template to amplify the open reading frame (ORF) of lacZ. DNA fragments of the integration site  were amplified from GS115. ADH1 terminator (TADH1) was amplified from the genomic DNA of S. cerevisiae BY4741. The fragment of ori and zeocin-resistant marker came from the commercial plasmid pPICZαC . All the fragments were amplified with Hieff Canace™ high-fidelity DNA Polymerase  and then be linked by the homologous arm with pEASY®-Basic Seamless Cloning and Assembly Kit  to construct a vector pZeocin/lacZ-TADH1.The genomic DNA of P. pastoris GS115 by PCR and then inserted into the HindIII site of pZeocin/lacZ-TADH1 with seamless cloning to yield vector pZeocin/PXXX-lacZ-TADH1 (PXXX indicates the name of candidate promoters).The promoter fragments of candidates were amplified from the genomic DNA of XXX-lacZ-TADH1 with PmeI (located in the middle of AOX1 promoter locus) digested were transferred into the GS115 cells by electro-transformation and integrated into the genome by homologous recombination [XXX-lacZ were selected from the agar plates containing zeocin (0.1 mg/mL) and verified by PCR. The template came from colony or genomic DNA. The colony was boiled in water for 5 min before PCR and the genomic DNA was extracted as the previous report [The linearized vector pZeocin/Pbination . The strs report .600 of 3) were collected. The supernatant was discarded by centrifuge, and the pellet was resuspended in 1 mL Z buffer  and diluted to make the final OD600 of 0.2–0.6. Take suitable volumes of the above cells into a 2 mL Eppendorf tube, and make up to 1 mL with Z Buffer. Add 25 µL 0.1% SDS and 50 µL chloroform, vortex for 30 s, and incubate at 30 °C for 15 min. The reaction was started through the addition of 200 µL of 4 mg/mL onitrophenyl-β-D-galactopyranoside (ONPG) solution at 30 °C and stopped by the addition of 500 µL of 1 M NaCO3. After removal of cell debris by centrifugation, the OD420 and OD550 of the reaction solution were detected by Evolution™ 220 spectrophotometer, and the relative activity of β-galactosidase was calculated using the formula below:The β-galactosidase activity was assayed according to the previous report with slight modification . BrieflycrtE-tHMG1 and vector pMRI-35-crtYB-crtI were the kind gifts from Hongwei Yu’s lab [tHMG1/crtE or crtI/crtYB containing the promoter, ORF, and terminator were amplified from the above vectors. The AOX1 promoter (located in GS115 chromosome 4) and ENO2 promoter (located in GS115 chromosome 3) were chosen as the integration site. The ble (coding zeocin-resistant protein in E. coli and yeast) and kan  were chosen as screening markers. All those fragments were linked with the homologous arm by seamless cloning to yield vector pZeocin/crtE-tHMG1 and vector pG418/crtYB-crtI.The vector pMRI-34-Yu’s lab . The two0107, PGCW14, P0019, PGAP, P0208, and P0230) were amplified from the genomic DNA of GS115 by PCR. Two promoters in opposite directions were co-amplified by fusion PCR. Then a pool of combinations with two reversed promoters was obtained. Those promoters were inserted into BamHI/NotI sites of vector pZeocin/crtE-tHMG1 and vector pG418/crtYB-crtI with seamless cloning to yield vector pZeocin/PXXX-crtE-PYYY-tHMG1 and vector pG418/PXXX-crtYB-PYYY-crtI (PXXX and PYYY indicate the name of candidate promoters). The vector pZeocin/PXXX-crtE-PYYY-tHMG1 was linearized with PmeI and then transferred into the GS115 cell by electro-transformation. All the transformants grown on the agar plates containing zeocin were selected and co-cultured for the next round of electro-transformation with linearized pG418/PXXX-crtYB-PYYY-crtI by ScaI in ENO2 promoter. The transformants with different colors on the agar plates containing G418 (0.25 mg/mL) were picked out. The promoter combinations were verified by PCR.The candidate promoters  equipped with a Supersil ODS2 C18 column (4.6mm × 250mm). The mobile phase is composed of acetonitrile, methanol, and isopropanol with a volume ratio of 5:3:2, followed by a flow rate of 1 mL/min at 40 °C. The UV/VIS signals were detected at 450 nm, and the analysis time of each sample is 40 min. The standard product of β-carotene was purchased from Solarbio . The standard curve of concentration with the peak area was verified by triplicate.The extraction and analysis of β-carotene were performed according to a previous report . BrieflyAdditional file 1. Identified promoter candidates in P. pastoris GS115.Additional file 2. Evaluation of the growth-dependent strong promoter P0208 andP0627. (A) The relative transcriptional levels of 0627, 0208,and 0407 in YPD broth (B) The relative transcriptional levels of 0627,0208, and 0407 in YPG broth (C) The relative transcriptional levelsof 0627, 0208, and 0407 in YPM broth.Additional file 3. Primers used for RT-qPCR and colony verification.Additional file 4. Sequence length of the intergenic region of selected genes.Additional file 5. Strains and vectors used in this study.