Xist) and is linked with the erasure of chromatin silencing. Over the past years, the advent of transcriptomics and epigenomics has provided new insights into the transcriptional and chromatin dynamics with which XCR takes place. However, multiple questions remain unanswered about how chromatin and transcription related processes enable XCR. Here, we review recent work on establishing the transcriptional and chromatin kinetics of XCR, as well as discuss a model by which transcription factors mediate XCR not only via Xist repression, but also by direct targeting of X-linked genes.Dosage compensation between the sexes results in one X chromosome being inactivated during female mammalian development. Chromosome-wide transcriptional silencing from the inactive X chromosome (Xi) in mammalian cells is erased in a process termed X-chromosome reactivation (XCR), which has emerged as a paradigm for studying the reversal of chromatin silencing. XCR is linked with germline development and induction of naive pluripotency in the epiblast, and also takes place upon reprogramming somatic cells to induced pluripotency. XCR depends on silencing of the long non-coding RNA (lncRNA) X inactive specific transcript ( X-chromosome reactivation (XCR) is a developmentally regulated process by which X-chromosome inactivation (XCI) is reversed ,2. It ortrans factors that promote transcriptional activation of the Xa . Chromatin silencing in cis  prevents reactivation by transcription factors and forms stable and heritable gene silencing. Although mouse pluripotent stem cells (PSCs) have been used as a key tool to study random XCI for decades, random XCI during the differentiation of human naive PSCs has only recently been reported [Previous work has revealed a strong link between pluripotency and XCR in placental mammals ,29,30,31reported . In linereported ,43. Partreported . In sum,Xist), a key inducer of XCI [Tsix, a lncRNA encoded antisense to Xist, is reactivated during XCR in mouse [Xist [During XCR, the long non-coding RNA (lncRNA) X inactive specific transcript , a region that is necessary and sufficient for XCI [Initiation of XCI is a remarkable example of developmentally regulated chromosome-wide heterochromatin formation in which over 1000 genes are silenced on the Xi . Althouge of XCI ,59,60,61 network , then sp network . Xist fonization a [62,63, for XCI ,68,69.Xist RNA triggers the recruitment of a plethora of proteins that induce, propagate and stabilize the silent chromatin state on the Xi  and RNA FISH have indicated that AK119ub1 , loss ofAK119ub1 , loss ofAK119ub1 , and chrAK119ub1 . Second,frequent ,93,96. Tgoes XCR , again s unclear . Fourth,the soma ,99. Such B cells ,115. HowA key mark for maintenance of XCI is promoter DNA methylation, which helps to maintain gene silencing especially in embryonic lineages ,102,103.Setdb1 has a very small but significant effect on maintenance of XCI [The repressive histone marks H3K9me2/3 have been shown to enrich on the Xi during XCI . Depletie of XCI , in linee of XCI . These re of XCI , but othEed maintains imprinted XCI by opposing XCR upon exit from the TE stem cell state [Dnmt1 knockout embryos, maintenance of XCI is unaffected in the extraembryonic endoderm while XCR takes place in the embryonic lineage [Eed in maintenance of imprinted XCI in the TE [Eed knockout had little impact on maintenance of XCI in the PE, indicating that distinct mechanisms maintain imprinted XCI in TE and PE lineages [Differences in the factors required to maintain XCI in distinct embryonic lineages have been reported. In the mouse TE, Polycomb group protein ll state ,105. Polll state ,126,127. lineage . A more n the TE . Howeverlineages . Given tOne key difference between the human extraembryonic and embryonic lineages is that embryonic lineages need to maintain stable chromatin silencing for up to over 100 years, while extraembryonic lineages support embryo and fetal development for less than a year. Hence, somatic and extraembryonic lineages might use distinct mechanisms for maintenance of chromatin silencing on the Xi, and on autosomes as well. Indeed, several studies have indicated that maintenance of XCI in somatic cells depends on a plethora of distinct mechanisms that include DNA methylation.Xist RNA together maintain gene silencing on the Xi [Xist has a much bigger effect on XCR than any individual condition alone. Collectively, these results suggest that multiple different layers of chromatin repression act together to maintain XCI and ensure long term maintenance of XCI. Studies in which multiple repressive pathways are targeted individually or in combination have established that synergism between DNA methylation, histone deacetylation and n the Xi . InhibitXist RNA, removal of repressive marks and recruitment of active marks on chromatin [Despite the presence of most of the repressive chromatin mechanisms that we know of on the Xi, stable chromatin silencing on the Xi can be reversed. There are two forms of XCR in mouse, reversal of imprinted XCI and reversal of random XCI. During mouse preimplantation stages of embryonic development, the transcriptionally repressed paternal X (Xp) is reversed in the epiblast, whereas the extraembryonic lineages TE and PE maintain imprinted XCI for further stages of the development ,2,26,33.hromatin ,2,129.Previous studies have revealed a strong link between pluripotency and XCR in placental mammals , but notNanog [Xist silencing [Xist silencing [RNA FISH and scRNA-seq experiments in early mouse embryos have shown that different genes are reactivated at different times during reversal of imprinted XCI. In preimplantation mouse embryos, XCR correlates with the expression of the pluripotency gene Nanog ,132,133.ilencing ,133. Howilencing . These rilencing . Indeed,ilencing . It is pilencing . Furtherilencing ,135,136.Nanog and Prdm14 [Xist silencing [Xist is silenced in most pre-migratory PGCs [Xic [The second round of XCR in mouse occurs in primordial germ cells (PGCs) and erases random XCI ,138,139.d Prdm14 , Xist siilencing , H3K27meilencing , and trailencing  . Moilencing . In addiory PGCs . XCR begGCs [Xic . In summSeveral in vitro systems have been used to induce and study XCR. These include cell fusion ,147,148,Tsix reactivation [Xist silencing [Reprogramming somatic cells into iPSCs erases memory of somatic cell identity and chromatin silencing ,152. RNAtivation ,25,42, Xilencing ,25,42, lilencing ,25,42, ailencing , DNA demilencing , and trailencing ,150,154.Dusp9 leads to repression of DNMTs and induces female-specific DNA hypomethylation [Klhl13 late in reprogramming likely stabilizes pluripotency and delays pluripotency exit in XX iPSCs [Xist silencing and erasure of XCI, in agreement with the strong link between pluripotency and the XaXa state, which is then responsible for inducing sex-specific molecular and cellular changes that are then lost upon acquisition of X chromosome aneuploidy.Multiple studies have also shown that the double dose of X-linked genes due to XCR in XX iPSCs has consequences for the molecular and cellular properties of iPSCs ,154,155,hylation ,158. XX hylation ,156. An XX iPSCs ,159. FurXX iPSCs ,156,158.Xist [Klhl13. Early reactivated genes also become highly expressed in the progression phase of XCR than in the initiation phase [Tsix mutation to force non-random XCI, an Hprt-GFP reporter, a Nanog reporter and an inducible reprogramming TFs cassette [Pluripotency induction in somatic cells leads to XCR ,25,42. AXist . Late reon phase . A more cassette . To deficassette . Partialcassette .Xist locus [Xist loss [Xist levels are already reduced during the initiation of XCR at the time early genes become bi-allelically expressed [Xist levels to maintain their silenced state during reprogramming to iPSCs. Because X-linked genes relocate to the interior of a repressive compartment during XCI, while escapee genes remain outside [Both studies found that early reactivated genes during iPSC reprogramming have a shorter genomic distance to escapee genes compared with intermediate and late reactivated genes ,49. Accost locus ,49. Althist loss , like inist loss ,129,133,xpressed ,49. Henc outside , it was  outside ,48,49. W outside . Collect outside ,163. If Xist silencing in a relatively short period of time. Finally, both studies use bulk measureements, and scRNA-seq will be needed to resolve the heterogeneity that is inherent to iPSC reprogramming.Finally, two studies, one in mouse embryonic fibroblast and the other in neural progenitor cells, reached different conclusions on how fast XCR takes place during reprogramming to induced pluripotency ,49. The We now know that the mammalian genome is organized hierarchically , and theDxz4 tandem repeat locus [Xist and H3K27me3, while the B-like compartment is enriched for heterochromatin-associated protein CBX1, also known as HP1-BETA [The Xi forms two large mega-domains delimited by the at locus ,168,169.at locus ,81,121. at locus . The A-lHP1-BETA . There iHP1-BETA ,81,121. HP1-BETA , it willtrans factors that are induced during reprogramming and increase the expression of early genes both on the Xa and on the Xi. Together, these results suggest that both chromatin opening and 3D chromatin organization might poise early genes for activation during iPSC reprogramming, possibly due to a combination of reduced Xist expression and TFs overexpression, all of which remains to be functionally tested. In summary, early reactivating genes are located adjacent to escapee genes, within an A-like compartment, and become partially open and transcriptionally reactivated early during XCR. Given that early reactivated genes during XCR have a shorter genomic distance to escapees ,49 and gXic, which contains Xist, also takes place during XCR [Xist silencing is key for XCR [Xist, Jpx and Ftx, whereas interactions between Ftx and Rlim increase [Jpx downregulation might facilitate Xist repression, while Ftx and Rlim are unlikely candidates for Xist repression due to their reactivation kinetics [Tsix, Xite and Linx (Xist repressors), occurs after XCR, suggesting that it may not be required for XCR. In summary, distinct changes in 3D-chromatin interactions take place at the Xic during the initiation, progression and completion of XCR.3D chromatin remodeling of the ring XCR . Xist si for XCR ,25. Earlincrease . Jpx dowkinetics . RestrucXist binding [XCR leads to the chromosome-wide reacquisition of TADs on the Xi . Indeed, binding . DiffereXic has long been suspected to play a key role in XCR [Xist [Xist and activate Tsix [The e in XCR ,68,69 beCR [Xist ,130. Pluate Tsix ,31,171. Xist silencing are needed for XCR to take place [Tet1 and Tet2 in addition to Tet3 depletion does not prevent Xi DNA demethylation [Smarrc1 and Smarca4, which were recently identified in a screen for factors involved in XCI [Additional mechanisms have been uncovered for erasure of Xi silencing. One study showed that the H3K27me3 histone demethylase UTX actively removes H3K27me3 on the Xi during XCR in the epiblast . Whetherke place ,51. DNA hylation . A key rd in XCI .Xic in inducing XCR is based on two key lines of evidence. First, Xist silencing is required for XCR [Xic, including the first intron of Xist, and the promoter of Xist [Tsix [Xist repression [Xic provides a simple and elegant mechanism linking pluripotency and XCI that also explains why loss of pluripotency triggers XCI. The proposed role of the  for XCR ,25. Seco of Xist . Third, st [Tsix . Fourth,pression ,40,172. Xic is the only regulatory region required for XCR. First, deletion of Xist intron 1 does not prevent XCR [Tsix, a repressor of Xist during development [Xist promoter with a tetracycline inducible promoter in the presence of inducer still leads to Xist repression during iPSC reprogramming, indicating that the promoter of Xist is not required for Xist silencing during reprogramming [Xist silencing and DNA demethylation are required for XCR [Xist deletion is not sufficient for XCR in most somatic cells and reprogramming to iPSCs [Xic is not required for maintenance of XCI [Xist silencing is necessary but not sufficient for XCR. Together, these results indicate that trans factors that also target other regulatory regions, outside the Xic as well, are likely important for reversing transcriptional silencing on the Xi. Clearly, more work is needed to understand the mechanisms underlying XCR and to identify the cis regulatory regions and trans factors involved. Still, there are several lines of evidence that also raise doubt whether the vent XCR . Second,elopment , is not elopment ,25. Thirgramming ,175. Fouto iPSCs ,112. Fife of XCI . TherefoXist but also bind across the Xi to mediate XCR. A key element of this model is that transcriptional activation requires TF binding; hence, TFs must at some point bind and activate transcription, but how this takes place during XCR remains to be determined.Recent studies have shown that TFs can act as pioneer factors to induce focal chromatin opening and DNA demethylation on autosomes during somatic cell reprogramming to pluripotency ,176. TheXic is involved in XCR. First, in somatic cells, Xist deletion is not sufficient to enable TFs involved in transcriptional activation on the Xa to bind to the Xi and induce transcription [Xist silencing is required. Second, multiple X-linked genes outside the Xic are bound by pluripotency TFs in ESCs [Several lines of evidence suggest that direct binding of pluripotency TFs to X-linked genes within and outside the cription ,112. It  in ESCs ,32,171. Xist expression, enabling early genes to be reactivated by pluripotency or other TFs that bind cooperatively with pioneer factors pre-bound to open chromatin regions. Upon further Xist silencing, repressive marks are lost, and pluripotency TF engage in focal binding on the Xi in key regulatory regions, concomitant with focal DNA demethylation and acquisition of chromatin accessibility. Then, spreading to other regions and binding of other TFs, including both pluripotency or other TFs already engaged in transcriptional activation on the Xa, takes place. This model would explain why a small category of genes reactivate before most other genes, and would also explain why transcriptional reactivation takes place upon Xist silencing and activation of the entire pluripotency gene regulatory network but not upon silencing of Xist in somatic cells. It also provides a mechanism by which Xist-independent chromatin marks would be reversed on the Xi.In this model, pluripotency TFs initially reduce Xist silencing, the mere removal of repressive marks by active or passive mechanisms could lead to binding by non-pluripotency-associated TFs. A key question is whether pluripotency TFs are involved at all in direct activation of Xi-linked gene expression during XCR, and whether they bind before or after other TFs. These questions remain to be addressed.The model above on Xi could be wrong, and after Xist during XCI [Recent work on transcription suggests that protein compartmentalization is likely involved in XCR. First, protein compartmentalization has just been implicated in the formation of stable gene silencing by ring XCI ,79,80. Tring XCI . TherefoXist expression is well correlated with H3K27me3 enrichment or gene silencing. Moreover, there is no imprinted XCI in humans [Although mice have been the main model species to study XCI and XCR, recent advances have enabled to also explore dosage compensation in humans. Expression analysis, RNA FISH and IF studies have demonstrated that in 4 cell stage human embryos, unlike in mouse, XIST is expressed and forms a cloud on both X chromosome in females and on the sole X chromosome in males ,179,180.n humans , hence, n humans . On the Single-cell RNA-seq analysis has confirmed bi-allelic expression of XIST from both X chromosomes in female and from the only X chromosome in male human pre-implantation embryos . FurtherXist is silenced. However, XIST is initially expressed on the Xa in the epiblast of early human embryos before silencing is induced, raising the question of whether XIST silencing is coupled to XCR in the human female germline. Although scRNA-seq analysis showed that female PGCs express XIST much more than male PGCs [During human postimplantation development, genome-wide reprogramming takes place in PGCs to prepare for the next generation. Mouse experiments indicated that the Xi undergoes XCR in female PGCs ,138,140.ale PGCs ,191, bulale PGCs . Moreoveale PGCs . A more It remains unclear whether XIST silencing is strictly correlated with XCR in the human germline. Collectively, the results suggest that XIST is expressed in female and male PGCs at a time when X-chromosome dosage is increased 1.6-fold in females over males. One possibility is that XIST is expressed in PGCs but does not silence, similar to pre-implantation human embryos, but different from mice. However, more work is required to answer questions such as: Does XIST form a dispersed RNA cloud as opposed to a compact RNA cloud in PGCs? Can XIST silence X-linked genes in female PGCs? Is XIST expression eventually silenced during PGCs development? scRNA-seq on human gonads as well as PGC-like cell differentiation protocols  could heWork over the past years by RNA-seq and RNA FISH has shown that, with a few exceptions, most human PSCs (both hESCs and human iPSCs (hiPSCs)) have one Xi that can undergo XCR by erosion of XCI ,197,198.Great efforts have been made to reprogram human primed PSCs as well as somatic cells to human naive PSCs, which also leads to XCR ,202,203.XCR is in fact considered one of the most stringent criteria used as a hallmark of human naive pluripotency ,183,208.XIST and XACT expression on the Xas in a small proportion of cells. Interestingly, naive cells derived from embryos possess a much higher proportion (30% of cells) with bi-allelic XIST expression [XIST silencing [Another two studies have considerably increased our understanding of XCR during primed to naive human pluripotency conversion ,200. Thepression ,200. Recpression . XIST reilencing . A smallilencing . WhetherEarly studies indicated that naive cells can undergo XCI upon differentiation, but XCI was non-random ,200. MorThe recent advent of improved developmental biology and cellular reprogramming approaches combined with genomic and gene editing technologies have provided paradigms for investigating the reversal of imprinted and random XCI. However, several unanswered questions remain .Answering these questions will help to better understand XCR, epigenetics, gene regulation, reprogramming and embryo development. Direct binding of pluripotency TFs during XCR remains a hypothesis which will need to be further investigated. Rapid advances in single-cell genomics and multi-omics as well as reprograming and gene editing and molecular and cellular biology will help to elucidate the reversal of gene silencing during development and reprogramming.


Drosophila. The mammalian Bucentaur/Craniofacial developmental protein 1 (Bcnt/Cfdp1) is also a tentative component of the SNF2-related CBP activator protein (Srcap) chromatin remodeling complex, but little is known about its properties, partly because few antibodies are available to examine the endogenous protein. In this paper, we assigned the Western blot signal against the mouse Bcnt/Cfdp1 as a doublet of approximately 45 kDa using anti-Bcnt/Cfdp1 antibodies, which were generated against either of two unrelated immunogens, BCNT-C domain or mouse N-terminal peptide, and in addition, the Cfdp1 knockdown mouse ES cell line and bovine tissue were used as potential negative controls. Moreover, LC-MS/MS analysis of the corresponding doublet to the Flag-tagged mouse Bcnt/Cfdp1 that was constitutively expressed in a HEK293 cell exhibited that the upper band was much more phosphorylated than the lower band with preferential Ser phosphorylation in the WESF motif of BCNT-C domain. Western blot analysis with these evaluated antibodies indicated a preferential expression of Bcnt/Cfdp1 in the early stages of brain development of mouse and rat, which is consistent with a data file of the expression of Bcnt/Cfdp1 mRNA.The Bucentaur (BCNT) protein family is characterized by a conserved amino acid sequence at the C-terminus (BCNT-C domain) and plays an essential role in gene expression and chromosomal maintenance in yeast and  Drosophila have been shown to play essential roles in gene expression and chromosomal maintenance .The BCNT family members in yeast and ntenance . Mammalintenance . This prntenance . Althougntenance . Swc5 isgulation , )Although Western blot analysis is much more powerful to examine the target proteins of extracts than mass spectroscopy analysis, these tools are fundamentally different—that is, individual Ab is not a tool to identify a molecule but rather a tool for checking inconsistencies such as with differential tissue distribution of mRNA expression. The specificity of an Ab depends largely on the relative expression level of the target molecule. Therefore, it is critical to understand the efficacy and limitations of the Ab used in any experiments.Recently, mass analysis tools have become remarkably advanced and widely available. Thus, it is now much easier to identify molecules that have been considered to be false targets due to nonspecific cross-reaction with Abs. These trials to identify the off-targets may provide byproducts of the excellent Abs, such as conformation-specific antibodies against the proteins.Bcnt/Cfdp1 in HeLa cells [As a more reliable anti-Bcnt/Cfdp1 Ab (e.g. A305-624A-M) becomes available, it is then possible to more accurately characterize endogenous Bcnt/Cfdp1, including subcellular localization and tissue distribution. On the other hand, the comparative transcriptome analysis of Cfdp1-K1 cells revealed that only a 25% decrease in Bcnt/Cfdp1 mRNA resulted in a marked up-regulation or down-regulation of many genes (Supplementary Tables S2 and S3). The finding is consistent with a report by Messina that suggests that extensive effects on vital cellular functions were caused by RNAi-depletion of La cells .Swc5 that does not encode the DEF/Y motif in the N-terminus [Recently, the budding yeast Swc5 has been clearly shown to preferentially bind to Histone H2A via the tandem DEF/Y motifs in the N-terminus , while aterminus . To furtClick here for additional data file.Click here for additional data file.Click here for additional data file.


Arabidopsis thaliana.We review recent progress in our understanding of genetic and molecular mechanisms that control flowering in  Arabidopsis thaliana.Many plants synchronize their life cycles in response to changing seasons and initiate flowering under favourable environmental conditions to ensure reproductive success. To confer a robust seasonal response, plants use diverse genetic programmes that integrate environmental and endogenous cues and converge on central floral regulatory hubs. Technological advances have allowed us to understand these complex processes more completely. Here, we review recent progress in our understanding of genetic and molecular mechanisms that control flowering in  Arabidopsis thaliana, flowering is promoted by distinct environmental cues, such as daylength (photoperiod), winter , and high ambient temperatures, as well as endogenous cues, such as plant age (ageing), the phytohormone gibberellin (GA), and the carbohydrate status , GIGANTEA (GI), CRYPTOCHROME2 (CRY2), FLOWERING LOCUS E (FE), CONSTANS (CO), and FLOWERING LOCUS T (FT)  zinc family which contains two N-terminal B-boxes and a C-terminal CONSTANS, CONSTANS-LIKE, TIMING OF CAB EXPRESSION1 (TOC1) (CCT) DNA-binding domain  Strayer.CO is light dependent and controlled through the formation of a complex between the ubiquitin ligase FKF1 and GI in late afternoon   but not in SDs  . AlthougCO and FT, FKF1 also increases the protein level of CO by inhibiting functional COP1 homodimerization  and SUPPRESSSOR OF PHYTOCHROME A-105 (SPA) (CO mRNA produces the CCT-truncated variant COβ that promotes HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1), a RING-finger-containing E3 ubiquitin ligase, and COP1-dependent proteasomal turnover of the full-length protein COα, whereas COβ is resistant to the activity of these E3 ubiquitin ligases , a central component of the circadian clock, whereas the related family members TOC1/PRR1, PRR5, and PRR7 engage in interactions with CO mainly in the late afternoon  in the promoter of fternoon   and TEM2 act in the early developmental stage to block floral transition. Thus, an important mechanism for FT regulation is the balance between CO and TEM genes  under low ambient temperatures , and SCHLAFMÜTZE (SMZ), also contribute to the repression of flowering under inductive and non-inductive photoperiod conditions (FT was shown only in plants overexpressing SMZ or TOE1 (A morning-specific inhibition of CO function occurs through an interaction with the miR172-sensitive APETALA2 (AP2)-type transcriptional regulator TARGET OF EAT1 (TOE1), whereas FKF1 relieves this repressive constraint by binding TOE1  component CURLY LEAF (CLF), a methyltransferase that catalyses the deposition of histone H3 lysine 27 tri-methylation (H3K27me3), one of the repressive marks, and is associated with gene silencing (FT and orchestrates recruitment of PRC components before dusk to mediate H3K27me3 deposition on FT chromatin (FT is sustained by the activity of LIKE HETEROCHROMATIN PROTEIN1 (LHP1) which binds to H3K27me3 sites in FT through its chromodomain , thus preventing PcG-mediated silencing of FT  (REF6 and the homologous genes EARLY FLOWERING 6 (ELF6) and JMJ13 have redundant functions; however, REF6 plays the major role in shaping the genome-wide distribution of H3K27me3 (Overexpression of RELATIVE OF EARLY FLOWERING 6 (REF6), a Jumonji (JMJ) domain-containing histone H3K27me3 demethylase, activates transcription of FT is linked to the EMF1-interacting H3K4me2-specific demethylases JMJ14, JMJ15, and JMJ18  in a mutually exclusive manner , which binds to a distal site in the FT promoter, to catalyse the removal of H3K36me2/3 at FT and thus regulates the proper timing for reproduction  (FT-related gene TERMINAL FLOWER1 (TFL1), which is a key floral repressor, interacts with the unphosphorylated form of FD via 14-3-3 proteins. Moreover, it has been suggested that the transcriptionally inactive ternary FD–14-3-3–TFL1 complex represents the ground state at the SAM  SYNTHASE 1 (TPS1) and T6P, which has been suggested to function as a signalling molecule of sugar status in plants, are also likely to contribute to the reduction in miR156 abundance , while BRM antagonizes mainly the function of SWN at the juvenile phase  promotes H2A.Z incorporation to facilitate ARABIDOPSIS TRITHORAX-RELATED7 (ATXR7)-dependent active chromatin formation at MIR156a/c  and TARGET OF FLC AND SVP1 (TFS1)  and GA3ox2 (SHORT VEGETATIVE PHASE (SVP) and FLC control GA metabolism through the regulation of GA20- and GA2-oxidases  complexes is disrupted through physical interactions between DELLAs and NF-Ys, thus suppressing SOC1 gene activation and the floral response in Arabidopsis , which suppresses GA signalling and promotes CK responses , and CYTOKININ-RESPONSIVE GATA FACTOR1 (CGA1)/GNC-LIKE (GNL) are downstream factors of GA and CK signalling and involved in a cross-repressive interaction with SOC1 to regulate floral and greening response .Table S3. List of the genes that regulate the transcript level of transcription factors.Table S4. List of the genes that regulate the function of transcription factors.Table S5. List of the genes that regulate flowering via epigenetic control.Table S6. The rest of the genes for flowering time control.eraa057_suppl_supplementary_tables_S1_S6Click here for additional data file.


Fibrosis remains a major unmet medical need as therapeutic targets discovered in animal models have failed to translate. A major challenge for identifying novel targets is the limited availability of early-stage human disease tissue. Here, we utilize Dupuytren’s disease (DD), a common localized fibrotic disorder, to evaluate the impact of epigenetic regulation of myofibroblasts and identify potential tractable targets in human fibrosis. We demonstrate that the epigenetic regulator CREBBP/EP300 is a critical determinant of the profibrotic phenotype. Furthermore, we identify collagen VI to be a key downstream target of CREBBP/EP300 and reveal valuable insights in the role it plays in key profibrotic functions, including contractile force, chemotaxis, and wound healing, and hence its potential as a therapeutic target. ACTA2 and COL1A1. Genomic analysis revealed that EP300 is highly enriched at enhancers associated with genes involved in multiple profibrotic pathways, and broad transcriptomic and proteomic profiling of CREBBP/EP300 inhibition by the chemical probe SGC-CBP30 identified collagen VI (Col VI) as a prominent downstream regulator of myofibroblast activity. Targeted Col VI knockdown results in significant decrease in profibrotic functions, including myofibroblast contractile force, extracellular matrix (ECM) production, chemotaxis, and wound healing. Further evidence for Col VI as a major determinant of fibrosis is its abundant expression within Dupuytren’s nodules and also in the fibrotic foci of idiopathic pulmonary fibrosis (IPF). Thus, Col VI may represent a tractable therapeutic target across a range of fibrotic disorders.Fibrotic diseases remain a major cause of morbidity and mortality, yet there are few effective therapies. The underlying pathology of all fibrotic conditions is the activity of myofibroblasts. Using cells from freshly excised disease tissue from patients with Dupuytren’s disease (DD), a localized fibrotic disorder of the palm, we sought to identify new therapeutic targets for fibrotic disease. We hypothesized that the persistent activity of myofibroblasts in fibrotic diseases might involve epigenetic modifications. Using a validated genetics-led target prioritization algorithm (Pi) of genome wide association studies (GWAS) data and a broad screen of epigenetic inhibitors, we found that the acetyltransferase CREBBP/EP300 is a major regulator of contractility and extracellular matrix production via control of H3K27 acetylation at the profibrotic genes,  Fibrosis of visceral organs such as the lungs, heart, kidneys, and liver is a major cause of morbidity and mortality . HoweverDupuytren’s disease (DD) is a debilitating fibroproliferative disorder restricted to the palms of genetically susceptible individuals . The iniDespite the substantial heritability of 80%  in DD anWe hypothesized that specific targeting of transcriptional coactivators could provide valuable insight into epigenetic mechanisms controlling the myofibroblast phenotype in fibrosis. Here we show that the histone acetyltransferases CREBBP and EP300  are key CREBBP and EP300 member genes, as shown at the intersection of delineated pathways in blue and green, respectively , providing unbiased support for our hypothesis that epigenetic modulators are important in controlling disease processes in DD.To leverage the power of genetic variance in target discovery, we applied our recently developed prioritization “Pi” approach  to risk SI Appendix, Table S2) to study their effect on the expression of key profibrotic genes  on early passage myofibroblasts derived from nodules in patients with DD . This revealed the pan bromodomain inhibitor bromosporine (BrSp), the BET bromodomain inhibitors, PFI-1 and JQ1, and the two structurally distinct CREBBP/EP300 bromodomains inhibitors SGC-CBP30 (referred to as CBP30 throughout) and I-CBP112, as the most potent and significant inhibitors of ACTA2 and COL1A1 gene expression . We also employed a recently developed, highly specific CBP/EP300 HAT inhibitor A485 .For functional validation, we screened a focused library of 39 epigenetic inhibitors  qPCR with antibodies for the H3K27ac mark. We also included the BET inhibitor JQ1 as it also reduced expression of ACTA2 and COL1A1 in our primary qPCR screen. Both CBP30 and JQ1 treatment reduced H3K27 acetylation at the transcriptional start sites of ACTA2 and COL1A1, confirming a direct role in regulating gene transcription , with JQ1, but not CBP30, also reducing cell viability and confluency . Myofibroblasts display spindle or stellate-cell morphology, and while treatment with CBP30 had no clear effect on myofibroblast morphology, JQ1 treatment also altered cell shape, resulting in an elongated neuron-like morphology, indicative of broad effects not shared with CBP30 . Collectively, these data indicate CREBBP/EP300 is a highly associated pathway in localized human fibrosis that controls key aspects of myofibroblast phenotype and function, including ACTA2 and COL1A1 gene expression and cell contractility.Since excessive myofibroblast contractility is a key feature in disease pathogenicity in vivo, we measured the effects of JQ1 and CBP30 on myofibroblast force generation as a function of contractility using traction force microscopy . This deTranscriptional regulation of gene expression is achieved through cooperation between promoter and enhancer elements. Specific histone modifications function as binding elements for effector proteins that serve to regulate transcription through manipulation of the chromatin environment or assembly of transcription machinery. Acetylation at lysine 27 of histone H3 serves as a signature mark of active enhancers  in all cRELA and AKT1 identified EP300 binding within 50 kB of the annotated genes. We have shown two genes each, FOXO3 and EGFR from the FoxO pathway and JUNB and CSNK1A1 from the WNT pathway in SI Appendix, Fig. S3, as an illustration of these findings.We next probed the ChIP sequencing (ChIP-Seq) datasets to verify EP300 binding within the genomic loci of all of the genes identified within the FoxO (blue) and WNT (green) pathways, previously identified from the Pi pathway analysis in ACTA2, COL1A1, and FN1 , both of which are highly associated with fibrosis in multiple organs . This demonstrated that knockdown of FOSL1 significantly increased expression of several genes, including CXCL8, IL6, and CTGF  and inhibited CCL26, SFRP4, and COL6A3 expression . In contrast, TEAD4 knockdown inhibited mRNA expression of CXCL8 and CCL2 , while RUNX1 knockdown significantly down-regulated mRNA expression of genes involved in ECM remodeling, including fibronectin-1 (FN1) and hyaluronan synthase 2 (HAS2) . Together, these data support the role of EP300 in modulating diverse profibrotic pathways and identified RUNX1, TEAD4, and Fra1 as EP300-associated transcription factors regulating discrete gene modules in myofibroblasts as summarized in To investigate a putative role for the EP300-regulated transcription factors in profibrotic gene expression, we performed siRNA-mediated gene knockdown of Fra1/Dataset S2). In contrast, bromodomain inhibition with CBP30 affected a much smaller set of genes  indicated that CBP30 down-regulated genes were significantly enriched in ECM and proteoglycan-associated protein pathways, integrin signaling, and syndecan-1 pathways (Dataset S3), in common with JQ1. Pathways unique to JQ1 included the Aurora A/B, PLK1, and FOXM1 pathways .Focusing on the most significantly enriched pathways, ECM and ECM associated, we performed cluster analysis of the data from six donors following bromodomain inhibitor treatment. The heatmap in SI Appendix, Table S5), most notable being the three collagen VI (collagen VIα1/2/3) isoforms in CBP30-treated cells (Dataset S4). We integrated the pathways generated by the transcriptomic dataset of CBP30-treated myofibroblasts with the proteome analysis to systematically explore the broader molecular landscape directed by CBP30 in myofibroblasts. This enabled a comprehensive CBP30 pathway analysis, which was found to be dominated by pathways associated with extracellular matrix organization . We also confirmed the presence of abundant levels of the full-length α3 chain of collagen VI in tissue culture supernatants of freshly isolated Dupuytren’s nodular cells and passaged MFs . These differentially expressed genes were associated with ECM remodeling, FOXM1 transcription network, integrin signaling, Syndecan-1, and the androgen receptor activity pathways (Dataset S6). This latter finding is of particular interest, given the male predominance of Dupuytren’s disease in younger age groups , suggesting this may be the dominant isoform driving the profibrotic function of collagen VI.As collagen VI was highly associated with the myofibroblast populations in human fibrosis, we further explored its function. We performed RNA-Seq on DD myofibroblasts treated with 4 or 6 d C 37). T. TCOL6A14 or 6 d D. This re groups . Syndecae groups . When coCOL6A1/2/3 depletion markedly reduces expression of multiple chemokines, we confirmed that COL6A1/2/3 siRNA treatment significantly inhibited CCL2 and CCL7 protein secretion by myofibroblasts . We confirmed that COL6A1/2/3 depletion in myofibroblasts significantly reduced chemotaxis of the myeloid cell line THP-1 toward myofibroblast conditioned media, suggesting collagen VI may play an important role in promoting immune cell recruitment in localized fibrosis  were also regulated by PCSK7 to a level similar to that observed by COL6A3 depletion alone .Proprotein convertases process and modulate proteins within the secretory compartment, at the plasma membrane and in the extracellular space  and, aloon alone J–M. PCSKClinical trials in human fibrosis have repeatedly failed, probably due to the poor predictive accuracy of targets identified using murine models of fibrosis. DD represents an important platform to study mechanisms of human fibrosis because of the ready availability of tissue from patients undergoing surgery as a therapeutic intervention. We combined multiomic profiling with functional validation using these well-characterized patient samples and identified collagen VI as a major regulator of the myofibroblast phenotype.50 of 1 μM, consistent with reported activity on the CBP-bromodomain  proposed CREBBP/EP300 as key epigenetic regulators of the myofibroblast phenotype in DD. Screening of a panel of high-quality epigenetic probes highlighted bromodomain inhibitors as potent regulators of profibrotic gene expression in DD myofibroblasts. While relying on chemical probes alone has pitfalls due to potential off-target effects, here we provide multiple lines of evidence of supporting a direct role of CBP/EP300 in profibrotic gene regulation. First, two structurally distinct bromodomain inhibitors CBP30 and I-CBP112, and the EP300 HAT inhibitor A485, display similar activity profiles in suppressing gene expression, with an ICmodomain , 43. SecH3K27ac and H3K4me1 occupancy are chromatin features used to identify cell-type-specific enhancers , 45. SigFOSL1) has previously been reported as a negative regulator of AP-1  transcriptional coactivators (CCL2 and CXCL8 (IL-8), important recruiters of monocytes and neutrophils. Depletion of RUNX1 highlighted a role of this transcription factor in the regulation of two key ECM regulators, fibronectin-1 (FN-1) and Hyaluronan Synthase 2 (HAS2). Together, these data describe a transcriptional network downstream of EP300 that orchestrates diverse cellular profibrotic functions in myofibroblasts associated with pathogenicity, including enhanced matrix remodeling, development of a contractile phenotype, and immunomodulatory properties, thereby providing a framework by which these transcription factors link EP300 gene regulation with the profibrotic phenotype typically associated with myofibroblasts.Our epigenetic profiling and motif analysis revealed several putative EP300 coregulators of gene transcription, including Fra1, RUNX1, and TEAD4. Functional analysis of these transcription factors demonstrated discrete functional properties of each in myofibroblasts. Fra1  in chemotaxis  and Ullrich congenital muscular dystrophy (UCMD) . A mouse defects . COL6A3  defects . Therefo defects , generat defects . However defects ; this wafibrosis . More reividuals . Given tividuals . FurtherA limitation of this study is the absence of appropriate control tissue. Dupuytren’s disease is restricted to certain fibers of the palmar fascia. Previous studies used cells from the fascia in the region of the carpal tunnel or the transverse carpal ligament from affected or normal individuals or uninvolved transverse palmar fibers from patients with Dupuytren’s disease as controls . HoweverIn summary, by exploiting the availability of samples from patients with DD and a combination of epigenetic and transcriptional profiling studies, we defined EP300 as an important regulator of human fibrosis that directs diverse cellular processes in myofibroblasts. There are supporting data showing that pharmacological inhibition of EP300 is effective in murine models of pulmonary fibrosis , 76. HowACTA2, COL1A1, COL3A1 and TGFB1 gene expression was performed using primary human myofibroblasts isolated from DD patients by Taqman PCR. Inhibition of acetylation at the promotors of ACTA2 and COL1A1 by the EP300 probe SGC-CBP30 (CBP30) was shown using ChIP PCR. Inhibition of myofibroblast contraction was assessed using traction force microscopy (COL6A1/2/3 depletion followed by RNA-Seq; subsequent roles in contraction, migration, and wound healing were confirmed using traction force microscopy, chemotaxis, and scratch assays, respectively. The methodology is described in detail in SI Appendix.In brief, screening the effects of a panel of epigenetic inhibitors upon croscopy . EvidencSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File


Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and is the causative agent of trachoma (infectious blindness) in disadvantaged populations. Over the course of its developmental cycle, Chlamydia extensively remodels its intracellular niche and parasitises the host cell for nutrients, with substantial resulting changes to the host cell transcriptome and proteome. However, little information is available on the impact of chlamydial infection on the host cell epigenome and global gene regulation. Regions of open eukaryotic chromatin correspond to nucleosome-depleted regions, which in turn are associated with regulatory functions and transcription factor binding. We applied formaldehyde-assisted isolation of regulatory elements enrichment followed by sequencing (FAIRE-Seq) to generate temporal chromatin maps of C. trachomatis-infected human epithelial cells in vitro over the chlamydial developmental cycle. We detected both conserved and distinct temporal changes to genome-wide chromatin accessibility associated with C. trachomatis infection. The observed differentially accessible chromatin regions include temporally-enriched sets of transcription factors, which may help shape the host cell response to infection. These regions and motifs were linked to genomic features and genes associated with immune responses, re-direction of host cell nutrients, intracellular signalling, cell–cell adhesion, extracellular matrix, metabolism and apoptosis. This work provides another perspective to the complex response to chlamydial infection, and will inform further studies of transcriptional regulation and the epigenome in Chlamydia-infected human cells and tissues.Chlamydia are Gram-negative, obligate intracellular bacterial pathogens responsible for a broad spectrum of human and animal diseases. In humans,  Chlamydia are Gram-negative, obligate intracellular bacterial pathogens responsible for a broad spectrum of human and animal diseases [Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection (STI) [Members of the genus diseases . In humaon (STI) , causingon (STI) , and is on (STI) . All memon (STI) . All chlon (STI) , 7. At ton (STI) .C. trachomatis infection alters host cell transcription over the course of its developmental cycle [NUclear Effector), a C. trachomatis type III secreted effector with methyltransferase activity, enters the host nucleus and methylates eukaryotic histones H2B, H3 and H4 in vitro [Bacterial interactions with mammalian cells can induce dynamic transcriptional responses from the cell, either through bacterial modulation of host cell processes or from innate immune signalling cascades and other cellular responses –11. In aal cycle  and may in vitro . HoweverC. trachomatis-infected HEp-2 epithelial cells and time-matched mock-infected cells, spanning the chlamydial developmental cycle . FAIRE protocols rely on the variable crosslinking efficiency of DNA to nucleosomes by formaldehyde, where nucleosome-bound DNA is more efficiently crosslinked. DNA fragments that are not crosslinked are subsequently enriched in the aqueous phase during phenol–chloroform extraction. These fragments represent regions of open chromatin, which in turn can be associated with regulatory factor binding sites. In FAIRE-Seq, libraries are generated from these enriched fragments, followed by sequencing and read mapping to a reference genome [Chlamydia-mediated effects from infection-specific or non-specific host cell responses. Further experiments with inactivated Chlamydia or selected gene knock-outs or knock-downs will help to elucidate the extent of specific Chlamydia-mediated interference with the host cell epigenome. We also note that the use of in vitro immortalised HEp-2 epithelial cells means that, despite their utility and widespread use in chlamydial research, the full diversity of host cell responses that are likely to be found within in vivo infections will not be captured.Genetic information in eukaryotes is compactly organised within the nucleus of each cell in highly ordered structures composed of DNA and proteins, designated chromatin. Cellular processes occur when chromatin fibres become less condensed, providing areas of open chromatin which allow transcription to proceed. Areas of open chromatin are associated with active DNA regulatory elements, including promoters, enhancers, silencers, and insulators. Chromatin accessibility is also relevant to alternative splicing, alternative promoter usage and alternative polyadenylation, where different forms of RNA are generated from the same gene . Thus, te genome , allowine genome . We idenC. trachomatis serovar E in sucrose–phosphate–glutamate (SPG) buffer as previously described [C. trachomatis EBs and mock-infected cell lysates were subsequently used to infect fresh HEp-2 monolayers. Fresh monolayers were infected with C. trachomatis serovar E in 3.5 mL SPG buffer for an MOI ~ 1 as previously described [2. Three biological replicates of infected and mock-infected dishes per time were harvested post-infection by scraping and resuspending cells in 150 μL sterile PBS. Resuspended cells were stored at − 80 °C.HEp-2 cells  were grown as monolayers in 6 × 100 mm TC dishes until 90% confluent. Monolayers were infected with escribed . Additioescribed . C. tracescribed , which rFormaldehyde-crosslinking of cells, sonication, DNA extraction of FAIRE-enriched fractions and Illumina library preparation was performed as previously described . Librarihttps://github.com/reganhayward/Manuscripts-code.Raw sequencing reads were trimmed and quality checked using Trimmomatic (0.36)  and FastPeak calling of open chromatin regions was performed using MACS2 (2.1.1)  in paireConsensus peak sets were created by combining significant peaks from the infected and mock-infected replicates for each time using Diffbind . Peaks wAnnotation of the set of differential chromatin-accessible regions was performed with Homer (v4.9)  and sepaResults within this manuscript show that 49% of the differently chromatin-accessible regions enriched in this chlamydial infection data are intergenic. Additional prediction-based software analyses would likely reduce the number of intergenic regions by predicting additional features such as silencers, insulators and possibly more enhancers. Although extremely useful in the right setting, we chose not to run prediction-based tools as we wanted the results to be less speculative and only highlight significant infection-relevant events.Bimodality coefficients for the frequency distribution plots were calculated using the R-package Modes . Long nop value < 0.001 and > 5% of target sequences. For the 120 conserved regions across all times, TFs were filtered based on a p value < 0.05. For significant de novo TFs, motif matrices were compared against the Jaspar [Motif analysis was performed with Homer . Target e Jaspar  and TomTe Jaspar  databaseC. trachomatis serovar E in HEp-2 cells over time). For infection relevance, TF expression was also examined across a range of different times , cell lines  and across different C. trachomatis-based infection models [All identified TFs from the conserved and time-specific regions were compared to publicly available gene expression data to ensure that each TF could be expressed in HEp2 cells . Following initial quality control measures, a single C. trachomatis-infected replicate was identified as an outlier and was removed from further analysis. Significant peaks, representing regions of open chromatin in either the mock-infected or infected conditions, were identified from reads mapped to the human genome; with 52,584,839 mapped reads for mock-infected replicates and 98,802,927 mapped reads for Chlamydia-infected replicates  . The resulting set of differential chromatin-accessible regions identifies both open and closed chromatin (relative to mock-infection). The total number of significant differentially accessible regions rose over the development cycle, with the number of regions increasing (3.6×) from 1 hpi (864) to 48 hpi 3128) , compared to the other times . The number of mapped reads was similar for all 12-h replicates across conditions, and similar to other times, suggesting minimal bias from the variability of the underlying mapped reads , cell defence , and cell stress/apoptosis . ATP7A, which has a promoter exhibiting an increase in chromatin accessibility, is a key regulator of copper transport into phagosomes as part of a host cell response to intracellular infection [The proportion of all differentially accessible regions mapping to promoter regions is 4 (0.5%) at 1 hpi, 14 (4.8%) at 12 hpi, 21 (1.5%) at 24 hpi and 265 (8.5%) at 48 hpi  and oligosaccharyltransferase complex subunit (OSTC), respectively  appeared at more than one time point post-infection. All genes exhibited changes in chromatin accessibility at intronic regions, except DNAJB5, which exhibited an increase in chromatin accessibility at its promoter and TTS. DGKB was the only gene to exhibit changes in chromatin accessibility at its promoter and at more than one intronic region, each with decreased accessibility. DGKB is a diacylglycerol kinase that metabolises 1,2-diacylglycerol (DAG) to produce phosphatidic acid (PA), a key precursor in the biosynthesis of triacylglycerols and phospholipids, and a major signalling molecule [Chlamydia obtains and redirects host-derived lipids through multiple pathways [Genes in immediate proximity to the promoter-specific differentially accessible chromatin regions were compared to genes containing differentially accessible intragenic peaks Fig. c. Of themolecule . Chlamydpathways .SDHB and CDHC), cell protection (IER3) and the stress-regulated polyubiquitin gene UBC (ubiquitin C) [SGK1 at 1, 12 and 48 h), with an increase in chromatin accessibility. SGK1 (serum/glucocorticoid regulated kinase) is associated with a range of different cellular processes that are crucial to reproductive activities, with deregulation resulting in reproductive disorders such as pregnancy loss, infertility and endometriosis [Changes in chromatin accessibility of regions overlapping tissue-specific transcriptional enhancers from a range of online databases were examined, identifying 316 enhancer and 13 “super-enhancer” regulated genes Fig. a, b. Theuitin C)  that is etriosis .Fig. 5Di“long-chain fatty acid biosynthetic process (GO:0042759)” with the greatest significance . Decreased availability of long-chain fatty acids such as lauric acid and capric acid can inactivate C. trachomatis [The majority (78%) of enhancer regions were found at 48 h post-infection, and predominantly exhibited decreased chromatin accessibility Fig. c. GO anachomatis . Reducedchomatis .CROCCP2, LA16c-321D4.2, LINC00514 and RP5-1173A5.1) by comparing chromatin accessibility changes that mapped to lncRNAs affecting cell growth and proliferation in HeLa cells [2FC ranging between − 3.3 and − 4.6). Due to the relative lack of annotation of many lncRNAs, assigning function bioinformatically is challenging. However, these lncRNAs have been found to directly impact the survival of HeLa cells [Four putative enhancer-regulated genes were further identified  and one within a promoter region (RPL27A). Only 5 of these 58 significant differentially accessible regions show a decrease in overall chromatin accessibility. However, these same regions also exhibit increased chromatin accessibility at different intragenic locations at 48 hpi, further highlighting the potential for infection-related alternative splicing mechanisms  are known transcriptional regulators [C8B at 1, 12 and 24 h and CFHR5 at 24 and 48 h). Conversely, C6 exhibits decreased chromatin accessibility at 48 h.The differential chromatin-accessible regions that are present at all four times during infection demonstrate a conserved host cell response to chlamydial infection Fig. b. Time-ssms Fig. a. The relication ; moreovegulators –77. We aETS1, POU3F2, TFAP4 and PKNOX1), represented by putative motifs within 49 different intergenic and intragenic regions. An increase in chromatin accessibility was seen at all binding sites and across all time points. TFAP4 (Transcription Factor AP-4) functions as an activator of gene-expression of both cellular and viral genes during cellular differentiation [ETS1  also functions as an activator and is able to directly control expression levels of cytokine and chemokine genes [C. trachomatis-based infection settings, as described in the methods  motifs in order to identify any potential master regulators of infection responses Fig. b. Four Tntiation . ETS1 , giving a broad visual representation of known biological events , transcription and translation , the cell cycle  and BCL2L14 (apoptosis facilitator Bcl-2-like protein 14) a member of the Bcl-2 Family of proteins that are linked to apoptosis [RPPH1, RN7SK, RN7SL2 and RMRP) that are involved in RNA processing, signalling and transcriptional regulation [SDCCAG8 and ORC2), and ubiquitination (PJA2 and FBXO46)  and energy production  , which is linked to a wide range of cellular functions including growth, proliferation and apoptosis. ErbB transmembrane receptors are also often exploited by bacterial pathogens for host cell invasion [C. pneumoniae Pmp21 as an EGFR-dependent mechanism of host cell entry [C. trachomatis Pmp21 ortholog, PmpD, also has adhesin-like functions [C. trachomatis inclusions, with calcium mobilisation and F-actin assembly disrupted [C. trachomatis attachment and development.The large number of genes associated with differential chromatin accessibility at 48 h permitted GO enrichment analysis to be performed, with the underlying genes distinguished by increased chromatin and decreased chromatin accessibility Fig. g. Signifinvasion . Notablyll entry . The C. unctions , howeverisrupted , indicatcell–cell adhesion via plasma membrane adhesion molecules’ . Several genes common to these categories with infection-responsive differential chromatin accessibility are associated with cadherins , which are calcium-dependent transmembrane glycoproteins associated with the actin cytoskeleton and an essential structural component to maintain cells bind together [C. trachomatis infection, and is linked to the alteration of adherens junctions and the induction of epithelial–mesenchymal transition (EMT) events that may underlie chlamydial fibrotic outcomes [SNX16 (see above), suggesting that alteration or disruption of cadherin regulation is a key feature of chlamydial infection. The lipid-based ontology ‘Membrane lipid biosynthetic process (GO: 0030148)’ was also associated with regions of open chromatin. Chlamydia scavenges a range of host cell-derived metabolites for intracellular growth and survival, particularly lipids [Three enriched biological processes share the term ‘together . Disruptoutcomes , 91. Alty lipids , 93.I-Smad (inhibition of Smad) binding (GO:0070411)’. I-Smads (inhibitory-Smads) are one of three sub-types of Smads that inhibit intracellular signalling of TGF-β by various mechanisms including receptor-mediated inhibition [‘Type 1 transforming growth factor beta receptor binding (GO: 0034713)’. In addition, four genes  are associated with closed chromatin and ‘R-Smad binding (GO: 0005814)’, which are part of the R-Smad sub-family that regulates TGF-β signalling directly [Chlamydia-infected cells, provoking runaway positive feedback loops that generate excessive ECM deposition and proteolysis, potentially leading to inflammation and scarring [Significantly enriched ontologies associated with regions of decreased chromatin accessibility include the ‘hibition . This codirectly , 96. TGFscarring .‘Microtubule organising centre (GO: 0044450)’. Dynein-based motor proteins have been shown to move the chlamydial inclusion via the internal microtubule network to the MTOC (microtubule-organising centre); the close proximity to the MTOC is thought to facilitate the transfer of host vesicular cargo to the chlamydial inclusion [We also identify 11 genes localised within the cellular component nclusion .‘Ubiquitin-like protein ligase binding (GO: 0044389)’ and ‘Ubiquitin protein ligase binding (GO: 0031625)’ are involved in ubiquitination and protein quality control. The eukaryotic ubiquitination modification marks proteins for degradation and regulates cell signalling of a variety of cellular processes, including innate immunity and vesicle trafficking [Chlamydia, host cell ubiquitin systems can mark chlamydial inclusions for subsequent destruction [Chlamydia species, using secreted effectors and other proteins, are able to subvert or avoid these host ubiquitination marks for intracellular survival [Two similar ontologies fficking . The depfficking . In Chlatruction  and thersurvival , 101. OuIRF3 (Interferon Regulatory Factor) motifs are enriched at 1 hpi; IRF3 is a key transcriptional regulator of type I interferon (IFN)-dependent innate immune responses and is induced by chlamydial infection. The type I IFN response to chlamydial infection can induce cell death or enhance the susceptibility of cells to pro-death stimuli [Chlamydia [Sp1) is a zinc-finger TF that binds to a wide range of promoters with GC-rich motifs. Sp1 may activate or repress transcription in a variety of cellular processes that include responses to physiological and pathological stimuli, cell differentiation, growth, apoptosis, immune responses, response to DNA damage and chromatin remodelling [Putative TFs were identified from enriched motifs within all significant differential chromatin-accessible regions at each time post-infection  at 48 h, in addition to further KLFs at 12 , 24 h (KLF 10) and 48 h (KLF 4), when relaxing the initial filtering steps . KLFs are zinc-finger TFs in the same family as  tissues . Dysregu tissues . We findntiation . KLF6 isferation . Target feration . KLF3 istabolism , with extabolism . KLF9 an tissues . KLF9 isppressor  and regupoptosis .KLF9 and KLF10 share the co-factor Sin3A (SIN3 Transcription Regulator Family Member A) [HDAC1 and HDAC2. HDAC2 has increased chromatin accessibility at all four time points, and HDAC9 has increased chromatin accessibility at 1, 24 and 48 h, further supporting the potential for histone modifications to be a component of the host cell response to chlamydial infection, or to be targets of chlamydial effectors [Histone deacetylases (HDACs) modify the core histones of the nucleosome, providing an important function in transcriptional regulation , and manember A) , which iember A) . The Sinffectors .KLF3 is a strong transcriptional repressor; here, we see a range of fold-changes in the KLF3 regulated genes, with two of the three datasets examined showing only a slight decrease in mean expression  showing that all replicates contain significant peaks genome-wide (human genome) without any visual chromosomal bias.Additional file 3. Annotation of all significant peaks. Annotation of all the significant peaks, with tabs separating genomic features and fold-change regulation.Additional file 4. Conserved transcription factor expression. Motifs associated with each transcription factor (TF) as identified within the conserved regions. Genes associated with these regions were compared against relevant gene expression data to identify their level of regulation during infection. The TF POU3F2 was not able to be compared as the motif was only identified within intergenic regions that could not be overlapped. A)ETS1 TF. B)TFAP4 TF. C)PKNOX1 TF.Additional file 5. Time specific regions. The list of time-specific differential chromatin-accessible regions. It should be noted that some genes in these lists are repeated at each time due to multiple peaks occurring at an annotated interval, that enhancers can affect more than one gene, and single genes can be affected by more than one enhancer.Additional file 6. Complete list of motifs and transcription factors. The complete list of significant motifs and enriched transcription factors.Additional file 7. Time-specific transcription factor expression. Motifs associated with each transcription factor (TF) (Table A)IRF3 TF from 1 h. B)Homeobox TF from 24 h. C-K) Nine TFs identified at 48 h.F) Table  were ide


During oogenesis and fertilization, histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs) tightly regulate the methylation of histone H3 on lysine-4 (H3K4me) by adding and removing methyl groups, respectively. Female germline-specific conditional knockout approaches that abolish the maternal store of target mRNAs and proteins are used to examine the functions of H3K4 KMTs and KDMs during oogenesis and early embryogenesis. In this review, we discuss the recent advances in information regarding the deposition and removal of histone H3K4 methylations, as well as their functional roles in sculpting and poising the oocytic and zygotic genomes. We start by describing the role of KMTs in establishing H3K4 methylation patterns in oocytes and the impact of H3K4 methylation on oocyte maturation and competence to undergo MZT. We then introduce the latest information regarding H3K4 demethylases that account for the dynamic changes in H3K4 modification levels during development and finish the review by specifying important unanswered questions in this research field along with promising future directions for H3K4-related epigenetic studies. In mammals, fertilization of an oocyte by a sperm results in the generation of a totipotent embryo that can form all cell types of the embryonic and extraembryonic lineages. Before, during, and shortly after fertilization, both parental genomes are transcriptionally silent, and early embryonic events are controlled by maternal mRNAs and proteins that are stored during oocyte growth . AcquisiBefore ZGA, early embryonic development is controlled exclusively by maternal products accumulated during oocyte development. After the elimination of a subset of the maternal products, transcription is re-initiated and developmental control passes to the zygotic genome . This drGametes are highly differentiated cell types. Therefore, fertilization requires major epigenetic remodeling to reconcile the paternal and maternal genomes with the formation of a totipotent zygote . At the Ddx4, Gdf9, and Zp3 were used to drive the oocyte-specific expression of CRE recombinase in the developing oocytes of transgenic mice, starting from embryonic day-16 (oocyte cyst stage) (Histone methylation is tightly regulated by histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs), which add and remove methyl groups, respectively. KMTs and KDMs have specificities for their target lysine residues, as well as for the number of methyl groups they can add or remove. Some of these enzymes have been genetically knocked out in mice. Deletion often leads to prenatal or perinatal lethality. To overcome early lethality, selected promoter fragments of t stage) , postnatt stage) , and dayt stage) , respectH3K4me3 is a common histone modification at the transcription start site of actively expressing genes in eukaryotic cells . BecauseThe SET1/COMPASS histone methyltransferase complex is the primary enzyme that methylates histone H3K4 . While yCxxC-finger protein 1 , which recognizes both preexisting H3K4me3 and non-methylated DNA and engages in multivalent chromatin binding of the whole complex . Phenotype analysis results showed that CXXC1 is essential for proper meiotic crossover formation. The deletion of Cxxc1 causes a decrease in H3K4me3 levels from the pachytene to the MII stage and gene transcription disorder. These studies suggest that CXXC1-mediated H3K4me3 not only directly controls meiosis-specific chromatin behaviors as in yeast but also plays an essential role in regulating genes essential for the meiotic progression of spermatogenesis and oogenesis.During meiotic prophase, a diploid germ cell produces haploid gametes with two consecutive rounds of division. Unique chromosomal events occur during the prophase of meiosis I, including programmed double-strand breaks and genome-wide homologous recombination . Successtra8-Cre . While tCxxc1 knockout mice indicated that SETD1-CXXC1 is one of the major KMT complexes that mediate H3K4me3 deposition on chromatin in mouse oocytes  6.5–7.5], and no embryonic stem cell lines could be generated using the Setd1a null embryos. In contrast, Setd1b null embryos are growth retarded from E7.5 and die around E11.5  were generated and analyzed  type of chromatin configuration. The chromatin of fully grown germinal vesicle oocytes undergoes a transformation from the NSN to the surrounding nucleolus (SN) type when the follicles grow to the antral stage . SN typeGdf9-Cre was employed to knock out Cxxc1 in oocytes as early as the primordial follicle stage, the Cxxc1-null oocytes exhibited reduced GVBD and polar body 1 (PB1) emission rates during meiotic maturation than wild type oocytes  during the G2-M transition is required for both mitotic and meiotic divisions , 2016. Cy haspin . Furthery haspin . All theCXXC1 proteins are rapidly degraded in oocytes after meiotic resumption, remain undetectable at the MII stage, and reaccumulate after fertilization . The samCXXC1 depletion during meiotic division is of physiological importance. Chromatin undergoes extensive reorganization during oocyte meiosis and fertilization, transitioning from a relatively relaxed configuration during maturation to a highly condensed state in mature eggs and returning to the interphase state after fertilization . The degThere are three variants of histone H3 in mammals, namely H3.1, H3.2, and H3.3. H3.1 and H3.2 variants are deposited on chromatin during the S phase of the cell cycle because their deposition is DNA replication-dependent. In contrast, the H3.3 variant is expressed and deposited in chromatin throughout the cell cycle in a DNA replication-independent manner. Dynamic histone exchanges in the chromatin of the oocytic and zygotic genomes are essential for maintaining normal transcriptional activity. Particularly, histone modifications associated with active gene expression, such as H3K4me3, are enriched in H3.3. Accordingly, the deposition of H3.3 is correlated with transcriptionally active genes.Cxxc1-null oocytes—the H3.3 incorporation rates were significantly decreased when compared with those in the control oocytes. The newly translated H3.3 proteins underwent remarkable de novo lysine-4 trimethylation in wild type oocytes in a CXXC1-dependent manner, even when DNA replication and transcription activities were absent. Furthermore, DNase I digestion assay for assessing chromatin accessibility in oocytes revealed that the genomic DNA of CXXC1-deleted oocytes was more resistant to DNase I digestion than the DNA from wild type oocytes. These results indicate that SETD1-CXXC1-mediated H3K4 trimethylation is essential for maintaining proper chromatin configurations, which makes the genome accessible to protein factors that facilitate transcription during oocyte development.Related to the focus of this review, the replacement of H3 variants is also regulated by post-translational modifications of H3. H3.3 is enriched in H3K4me3, whereas H3.1 is enriched in dimethylated H3 lysine-9 (H3K9me2). In a histone replacement experiment—involving microinjection of mRNAs encoding Flag-tagged histone H3 variants into GV stage-arrested In mammals, the maternal and paternal genomes are not functionally equivalent; both the maternal and paternal genomes are required for embryonic development. The most distinguishing feature that differentiates the sperm genome from the oocyte genome is that it is globally compacted with protamine proteins rather than with histone proteins . HoweverShortly after the formation of the male and female pronuclei, H3K4me1 exhibits comparable levels in both pronuclei; however, H3K4me2 and H3K4me3 are initially present at lower levels in male pronuclei than in the female pronuclei. The acquisition of H3K4me2/me3 in the male pronucleus only occurs at the latest pronuclear stages . ResearcCxxc1-deleted oocytes can be fertilized, but none of the embryos developed beyond the 2-cell stage, indicating that the absence of maternal CXXC1 in oocytes affects the developmental potential of the resulting zygotes after fertilization , specifically catalyzes the demethylation of H3K4me1 and H3K4me2. Mammals contain two members of the KDM1 family, KDM1A and KDM1B . Both enzymes play crucial roles in mammalian oogenesis and MZT . KDM1A iKdm1a prior to gastrulation results in early lethality  directly interacts with KDM1A and phosphorylates it at Serine-126 . As a rede novo DNA methyltransferase (DNMT) 3A and its cofactor DNMT3-like (DNMT3L) play a direct role in this process —regulates the parental origin-specific expression of imprinted genes in mammalian genomes. Epigenetic imprints in male and female germ cells are established during gametogenesis and are maintained throughout development. The  process . In addide novo DNA methylation in some imprinted genes in the oocytes  embryos at the 4-cell stage, Kdm5b fails to be activated in 4-cell stage-arrested SCNT embryos. To determine the role of Kdm5b in the development of SCNT embryos, they deleted or overexpressed Kdm5b in SCNT embryos, and found that the developmental potential of these embryos was decreased and increased, respectively  domain . The KDM fertile , suggestectively . Co-injeectively . These rKdm5a and Kdm5b show peak expression in 2-cell embryos and are responsible for actively removing broad H3K4me3 domains. Embryos depleted for Kdm5a/b retain high levels of H3K4me3 at the late 2-cell stage. A significant number of ZGA genes were downregulated in Kdm5a/b-depleted 2-cell embryos. As a result, the Kdm5a/b-depleted embryos fail to develop to the blastocyst stage  is species-specific and occurs at the mid-1-cell stage in mice . A low lLastly, the findings described above indicate that the spatiotemporal-specific establishment and erasure of H3K4me3 during oocyte development enables the oocyte genome to establish the competence to generate a healthy embryo in a cell-autonomous manner. Nevertheless, the extent to which this epigenetic modification forms a cell non-autonomous instructive component of ovarian follicle development remains unclear. Recent studies have revealed that appropriate levels of H3K4me3 accumulation in growing oocytes are necessary to maintain the expression of genes encoding oocyte-derived paracrine factors, including growth and differentiation factor 9 (GDF9), bone morphogenic protein 15 (BMP15), and fibroblast growth factor 8 (FGF8) . CXXC1-dH-YF and Q-QS conceived and wrote the manuscript. JZ prepared the figures. All the authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacillus subtilis σA-dependent rRNA promoters in a growth phase-dependent manner. The more negative DNA supercoiling in exponential phase stimulates transcription from rRNA promoters, and DNA relaxation in stationary phase contributes to cessation of their activity. Novobiocin treatment of B. subtilis cells relaxes DNA and decreases rRNA promoter activity despite an increase in the GTP level, a known positive regulator of B. subtilis rRNA promoters. Comparative analyses of steps during transcription initiation then reveal differences between rRNA promoters and a control promoter, Pveg, whose activity is less affected by changes in supercoiling. Additional data then show that DNA relaxation decreases transcription also from promoters dependent on alternative sigma factors σB, σD, σE, σF, and σH with the exception of σN where the trend is the opposite. To summarize, this study identifies DNA topology as a factor important (i) for the expression of rRNA in B. subtilis in response to nutrient availability in the environment, and (ii) for transcription activities of B. subtilis RNAP holoenzymes containing alternative sigma factors.The expression of rRNA is one of the most energetically demanding cellular processes and, as such, it must be stringently controlled. Here, we report that DNA topology, i.e., the level of DNA supercoiling, plays a role in the regulation of  Bacterial cells need to adapt to environmental changes. In nutrient-rich environments, cells grow and divide rapidly, and this requires a large number of ribosomes to satisfy the need for new proteins. In nutritionally poor environments, the synthesis of new ribosomes stops. As the production of new ribosomes is energetically costly for the cell, it must be tightly regulated. The number of ribosomes in the cell is regulated mainly on the level of transcription initiation of ribosomal RNA (rRNA) .Transcription initiation can be divided into several steps. First, when the RNA polymerase (RNAP) holoenzyme (the core RNAP subunits α2ββ´ω] in complex with a σ factor) binds to specific DNA sequences, promoters, it forms the closed complex where DNA is still in the double-helical form ββ´ω in c,4,5,6. SIn bacteria, the concentrations of iNTPs act as key regulators of transcription and directly affect RNAP at some promoters. These promoters form relatively unstable open complexes where the time window available to iNTPs to penetrate into the active site and initiate transcription is relatively short. The higher the concentration of the respective iNTP, the higher the chance that it penetrates into the active site while the transcription bubble is still open. Hence, increases in intracellular concentrations of iNTPs stimulate transcription whereas low levels of iNTPs result in inefficient transcription initiation ,9,10.Bacillus subtilis, a model soil-dwelling, spore-forming Gram-positive bacterium, the iNTP of the tandem rRNA promoters of all 10 rRNA operons is exclusively GTP ) affects rRNA promoter activity under stringent conditions , B. subtilis rRNA promoters are regulated by the level of their supercoiling, and we dissect the effects of supercoiling on the formation of closed and open complexes, thereby providing mechanistic insights into the process. Finally, we show that supercoiled (SC) DNA is a more efficient template for transcription for all alternative σ factors tested with the exception of σN, a recently discovered sigma factor encoded on the pBS32 plasmid of the NCIB 3610 strain  KH2PO4 (100 μCi/mL) until early exponential phase (OD600 ~0.3). Samples were taken until 250 min after OD600 ~0.3 (time 0). Samples (100 μL) were pipetted into 100 μL 11.5 M formic acid, vortexed, left on ice for 20 min, and stored overnight at −80 °C  H3PO4 (100 μCi/mL), and at time 5 min treated with novobiocin (5 μg/mL). Samples were taken at time points 0, 5, 10, 20, and 30 min and processed in the same way as above.To determine the relative ATP/GTP concentrations after novobiocin treatment, LK134 was grown to ODlacZ and activities were assayed by primer extension of the short-lived lacZ mRNA that allows to observe rapid decreases in promoter activity in time. The experiments were conducted as described in  UTP.Transcription experiments were performed with the GTP determination experiments, the amount of DNA (SC or LIN form) was 100 ng, ATP, CTP were 200 μM; UTP was 10 μM and GTP was titrated from 0 to 2000 μM. To determine the affinity of RNAP to DNA, ATP, CTP were at 200 μM; UTP was 10 μM, GTP was 1000 μM and DNA (SC/LIN) was titrated from 0 to 900 ng per reaction. In reactions with alternative σ, DNA (SC or LIN form) was 100 ng, CTP were at 200 μM; UTP was 10 μM and GTP/ATP was 1000 μM, depending on the identity of the base in the +1 position of the transcript. In KAll transcription reactions were allowed to proceed for 15 min at 30 °C and then stopped with equal volumes of formamide stop solution . Samples were loaded onto 7 M urea-7% polyacrylamide gels and electrophoresed. The dried gels were scanned with Molecular Imager FX (Bio-Rad) and were visualized and analysed using the Quantity One software (Bio-Rad).rrnB P1 promoter as it is one of the best-characterized rRNA promoters in B. subtilis that is regulated by [iNTP], , ,34,35,36ormation ,38. PromlacZ fusions. The endogenous copy of Pveg initiates transcription with ATP (+1A). Here, we used a +1G variant of Pveg so that both transcripts (from rrnB P1-lacZ and Pveg-lacZ) were identical, excluding any effects due to, e.g., potentially differential decay of the transcripts. The +1G Pveg promoter variant behaves identically with the +1A variant ) and (ii) relative promoter activity  with novobiocin or mock-treated them, and measured the rates of total RNA synthesis by following incorporation of radiolabeled 3H-uridine into RNA. As a positive control, where we expected cessation of RNA synthesis, we treated cells with rifampicin, a well-characterized inhibitor of bacterial RNAP.In this experiment, we first used total RNA as a proxy for rRNA synthesis as in exponential phase most of RNA synthesis comes for rRNA operons . We rrnB P1 and Pveg to novobiocin treatment, using the same conditions as in the previous experiment. We grew cells carrying the appropriate fusions (rrnB P1-lacZ (LK134) and Pveg-lacZ (LK135)) to early-exponential phase (OD600 ~0.3) and either treated them with novobiocin or mock-treated them. In the case of rrnB P1, the promoter activity decreased after novobiocin treatment (as opposed to mock treatment), but in the case of Pveg, the promoter activity displayed the same moderate decline regardless of the novobiocin treatment, suggesting that rrnB P1 is more sensitive to changes in DNA topology : the GTP level does not change but the open promoter becomes less stable, requiring more iGTP for maximal transcription. To address this hypothesis experimentally, we performed in vitro transcriptions with defined components. We used promoter core variants of rrnB P1 and Pveg cloned in the pRLG770 plasmid  . The GTPGTP for rrnB P1 was 277 ± 24 μM, and for Pveg 36 ± 9 μM. Experiments with the LIN templates then revealed that KGTP values for both promoters increased . In the case of rrnB P1 the KGTP increased from SC to LIN ~1.5x, and in the case of Pveg KGTP ~14x. Surprisingly, the KGTP value of LIN Pveg was even higher than the value for rrnB P1  of the rrnB P1 promoter, we also decided to use an extended version of the promoter region to assess whether the surrounding sequence has significant effects. Therefore, we used a DNA fragment containing both rrnB P1 and rrnB P2 promoters in their native tandem arrangement. Each of them contained their respective native -60 to -40 regions encompassing the UP elements. UP elements are A/T-rich sequences that enhance promoter activity by binding the C-terminal domains of α-subunits of RNAP [B. subtilis is less pronounced than, e.g., in E. coli (~30x), it is still significant [GTP for rrnB P1 (from the tandem promoter fragment) was 242 ± 31 μM for SC and 361 ± 46 μM for LIN. KGTP for rrnB P2 was 62 ± 13 μM for SC and 427 ± 61 μM for LIN  must be involved in the decrease of this promoter’s activity in vivo. A likely candidate factor was the affinity of RNAP for promoter DNA, i.e., formation of the closed complex or/and the intracellular level of RNAP.Hence, we concluded that for transcription from LIN templates higher concentrations of GTP are needed, regardless of the promoter. The increased KrrnB P1+P2 DNA fragment and Pveg. The GTP concentration was set to 1 mM to ensure high efficiency of open complex formation for the tested promoters. Affinity for RNAP of both rRNA promoters was unchanged or slightly decreased on relaxed templates, but this effect was not statistically significant (rrnB P1 (SC vs LIN) in vitro could be due to yet another factor . We tested the relative affinity of RNAP for promoter DNA by performing in vitro transcriptions as a function of increasing promoter DNA concentration. We used the tandem nificant . Therefoveg: a relatively low level of the relaxed promoter DNA was able to saturate RNAP compared to the supercoiled template. This behavior could then explain why the activity of Pveg decreased less than the activity of rrnB P1 both in vitro and in vivo. Importantly, it was previously reported that the levels of RNAP subunits decrease from exponential to stationary phase [The opposite trend was observed with Pry phase ,51 and wB, σD, σE, σF and σH. σB is a general stress response sigma factor [D transcribes genes linked with the cell motility and flagella formation [E and σF are sigma factors of early sporulation [H is responsible for transcription of early stationary genes [To extend the study, we tested the effect of supercoiling on transcription from promoters dependent on alternative sigma factors: σa factor ,53, σD tormation . σE and rulation ,56. σH iry genes . N (ZpdN) that is present only in the B. subtilis NCIB 3610 strain. This strain possesses a large, low-copy-number plasmid pBS32, which was lost during domestication of the commonly used laboratory strains [N [We tested also σ strains . pBS32 crains [N ,25. MMC rains [N ,60,61. TA . This construct contains σA-dependent sigN P2 and σΝ-dependent sigN P3 promoters [A-dependent P2 is more active on SC DNA whereas σN-dependent P3 prefers LIN DNA for efficient transcription.The exception was σpromoter B. To shoromoters  and alloBacillus subtilis RNAP to transcribe from σA-dependent rRNA promoters as well as from selected promoters depending on alternative σ factors.In this study we have identified the supercoiling level of DNA as a factor affecting the ability of B. subtilis rRNA promoters in stationary phase was also observed in [In our experiments, the drop in rRNA promoter activity during transition to stationary phase was pronounced and concurrent with the onset of stationary phase. A decrease in the activity of erved in . HoweverB. subtilis rRNA promoters, revealing supercoiling as a factor involved in their control. The more negatively supercoiled DNA in exponential phase contributes to the high activity of B. subtilis rRNA promoters. As this negative supercoiling becomes more relaxed when the cell transitions into stationary phase, this likely contributes to the decrease in the activity of RNAP at rRNA promoters. This is in part due to the decreased affinity of RNAP at rRNA promoters for the initiating GTP but also to a so far unknown step during transcription initiation . The activity of rRNA promoters in stationary phase is also likely affected by the decreased RNAP concentration. The decrease in the available RNAP pool is further exacerbated by the association of the RNAP:σA holoenzyme with 6S-1 RNA that sequesters it in an inactive form in stationary phase [E. coli rRNA promoters, the decreased stability of the open complex was identified as the main kinetic intermediate affected by supercoiling [S. aureus in post-exponential growth phase the downregulation of rRNA is independent of ppGpp or NTP pools [Here, we propose an updated model of regulation of ry phase . The comrcoiling . We alsoTP pools , and it B. subtilis, correlations between the supercoiling level and rRNA activity could be found also in the forespore. Within the developing spore, DNA becomes more negatively supercoiled then in stationary phase [B. subtilis and the changes in DNA topology override its stimulatory effect so that the net result is a decrease in the activity of rrnB P1. This is the first observation of a situation where the GTP level and rRNA promoter activity do not correlate in B. subtilis. We note that supercoiling was also reported to be involved in rRNA expression in yeast although the mechanistic aspects of this regulation are less understood [In ry phase  and thisry phase . Interesderstood .veg promoter also decreases from exponential to stationary phase but the decrease is not as pronounced as in the case of rrnB P1. The decrease in the activity of Pveg can be attributed, at least in part, to its increased requirement for the concentration of the iNTP when DNA supercoiling relaxes. Nevertheless, the affinity of Pveg for RNAP seems to increase with DNA relaxation and this likely partially counteracts the negative effect on open complex formation.The activity of the control PB, σD, σE, σF, and σH). This trend was previously reported also for RNAP:σH transcribing from the spoIIA promoter [F, this is consistent with the DNA supercoiling increase in the forespore [H and σE that are active in stationary phase, although activities of respective promoters strongly decreased with reduced supercoiling in vitro, this likely reflects the physiologically relevant requirements for their activities in the cell in stationary phase. Also, the decrease in the level of supercoiling in stationary phase is likely not as extreme as in our in vitro experiments where it was used to better visualize the effects.Transcription experiments with promoters dependent on alternative σ factors revealed that linear templates are poorer substrates for the majority of them  is either relatively unaffected or even increased on linear templates. This is likely physiologically important as mitomycin, which induces σN expression [N may have evolved to be most active under such conditions. The proficiency of RNAP:σN on linear templates then may stem from the relatively short spacers of σN dependent promoters , analogemplates ,69,70.B. subtilis and reveal the effect of supercoiling on transcription with main and alternative σ factors. To conclude, our findings extend the current model of rRNA promoter regulation in