Transcription factors, also known as trans-acting factors, balance development and stress responses in plants. Branching plays an important role in plant morphogenesis and is closely related to plant biomass and crop yield. The apical meristem produced during plant embryonic development repeatedly produces the body of the plant, and the final aerial structure is regulated by the branching mode generated by axillary meristem (AM) activities. These branching patterns are regulated by two processes: AM formation and axillary bud growth. In recent years, transcription factors involved in regulating these processes have been identified. In addition, these transcription factors play an important role in various plant hormone pathways and photoresponses regulating plant branching. In this review, we start from the formation and growth of axillary meristems, including the regulation of hormones, light and other internal and external factors, and focus on the transcription factors involved in regulating plant branching and development to provide candidate genes for improving crop architecture through gene editing or directed breeding. Transcription factors (TFs), also known as trans-acting factors, are proteins with special structures that regulate plant growth and development. Transcription factors bind to specific DNA sequences (cis-acting elements) in the upstream promoter region of target genes through their DNA-binding domain (DBD), thereby regulating the specific expression of target genes in different cell types of plants or under different environmental conditions . In planShoot branching is a common phenomenon in plant growth and plays a very important role in plant morphogenesis. Branching also affects plant competitiveness against weeds or pests ,3. ThereOryza sativa, Arabidopsis thaliana, Lycopersicon esculentum, Zea mays and other plants. However, the nomenclature of homologous genes in different species is confusing, and related studies on transcription factors involved in the regulation of branching development are lacking in systematic elaboration. Branch formation is regulated at two developmental stages: axillary bud meristem formation and axillary bud emergence. The latter is induced by bud dormancy release and regulated by the synergistic effect of plant hormones such as auxin (IAA), strigolactones (SLs), cytokinins (CKs), abscisic acid (ABA) and brassinosteroids (BRs) [Through a study on the regulatory mechanism of plant branching, a series of branching-related transcription factors  have beeds (BRs) ,7. Of cods (BRs) .In this review, we begin with the formation and growth of axillary meristems to elaborate on the research progress of transcription factors involved in regulating plant branching development to provide target genes for manipulating plant branching.Arabidopsis, rice, maize and tomato. The AM is formed in the center of the frontal boundary zone of the leaf base. This region is not only a boundary but also plays an important role in the maintenance of meristem and organ development [The first step of branching is the development of axillary meristems in leaves. In recent years, a series of transcription factors that regulate the initiation of leaf axil meristems have been found in elopment .Arabidopsis (WUSCHEL (WUS) is a homologous domain transcription factor that is expressed in the center of the SAM and specifies the fate of meristem cells in this region. The wus mutation leads to an inability to maintain stem cell division ability [LAS expression by binding to its promoter, promoting AM initiation [WUS expression through ARR1, enabling stem cell differentiation and axillary bud formation [The origin of AMs is a controversial topic. There is a major view that AMs originate from meristem cell groups that become detached from the shoot apical meristem (SAM) as leaves form and never lose their meristem identity . Early Abidopsis , WUSCHEL ability . ARABIDOitiation ,13. Cytoormation .MERISTEMLESS (STM) is another important factor for maintaining branch organization. STM, a KNOX gene, is expressed in the whole SAM but is excluded from the organ primordium that maintains the function of undifferentiated cells in the SAM [STM in the center of the boundary region [STM is downregulated by the MYB family transcription factor ASYMMETRIC LEAVES1 (AS1) and the LBD family transcription factor AS2 [ the SAM . The moly region . Once cector AS2 . This inctor AS2 .STM expression and promotes AM initiation. Subsequently, CK reactivates WUS expression to establish the AM [STM and WUS and that Ls/LAS acts upstream of STM to activate expression [REV is regulated by LAS. As an HD-ZIP transcription factor, REV itself is necessary for all lateral meristem formation. In addition to RNA accumulation in other modes, REV is expressed in the near-axis position of the developmental leaf primordium in a region similar to RAX1, which can produce the position signal of RAX1 expression and control the radial mode [drn-1 mutant, the drn-1 drnl-1 double mutant shows more serious defects in axillary bud formation than the single mutant, indicating that drn and drnl have important redundant functions in AM initiation [STM expression by binding to the same promoter region. In summary, DRN and DRNL redundantly promote AM initiation at the vegetative growth stage, and DRN/DRNL and REV synergistically upregulate STM transcription in mature leaf axils [Before axillary bud formation, REVOLUTA (REV) upregulates h the AM . Prelimipression ,20. Howeial mode ,22. The af axils .rax1) and las mutations enhance these branching defects [RAX1 and LAS genes, thereby affecting the growth of axillary buds. RAX1 is a member of the largest MYB TF R2R3-MYB family in Arabidopsis. RAX2 and RAX3 genes function in the early stages of AM initiation and development [REGULATOR OF AXILLARY MERISTEM FORMATION (ROX) encodes direct homologs of bHLH transcription factors, namely, LAX1 and BA1. In these mutants, axillary bud formation during vegetative bud development is damaged, and their combination with REGULATOR OF AXILLARY MERISTEMS1  and LOF2 encode MYB transcription factors that play roles in lateral organ separation and axillary meristem formation, partly through interaction with CUC2, CUC3 and STM ,28. LOF1 and CUC ,29,30. Tf the AM .Arabidopsis SAM via STM expression [CUC2 is also regulated by RAX1 in early AM development to establish or maintain the stem cell niche formed by the AM [CUC2 is continuously downregulated in the raxl mutant, indicating that RAX1 affects AM initiation by regulating the expression of CUC2 [LAX1 and CUC [RFL is expressed in the vegetative axillary meristem and very young tillering buds, and its expression pattern is similar to that of STM, which may be related to the maintenance of the meristem cell zone [The NAC domain proteins CUP SHAPED COTYLEDON1 (CUC1), CUC2 and CUC3 are involved in the initiation of the pression ,22,27,28pression . Among tpression ,31,32. Fpression . CUC2 is of CUC2 . Thus, R and CUC . RFL is ell zone .EXCESSIVE BRANCHES1 (EXB1) gene, which encodes a WRKY transcription factor previously known as WRKY71 that is mainly expressed in tissues around the AM initiation site. The functional exb1-D mutant displays an obvious increase in branching, which is due to the combined effect of excessive AM priming and increased bud activity. Quantitative data show that EXB1 controls the initiation of the AM by positively regulating the transcription of RAX1, RAX2 and RAX3. Subsequent data indicate that EXB1 may be located upstream of the RAX gene and regulate AM formation [Arabidopsis through negative regulation of auxin signaling [WRKY72 in Arabidopsis also increases bud branching, suggesting that the role of EXB1 in bud branching may be evolutionarily conserved between monocots and dicots [Arabidopsis is the transcription factor LATERAL ORGAN BOUNDARYES1 (LOB1), which induces PHYB ACTIVATION TAGGED SUPPRESSOR1 (BAS1) expression and encodes a protein that brassinosteroid-inactivation capacity. In leaf axils, BR accumulation is negatively regulated by LOB1, an important boundary-specific transcription factor [BAS1 to produce low BR concentrations to reduce cell division and expansion in border areas [Arabidopsis [Guo et al.  identifiormation ,36. EXB1ignaling . Auxin iignaling ,38. In tignaling . WRKY prd dicots . Furthern factor . LOB1 dier areas . Unlike bidopsis .TILLERS ABSENT1 (TAB1) and WUSCHEL-RELATED HOMEOBOX4 (WOX4) are two WUS genes that are necessary for AM initiation [TAB1 is normally expressed only in the anterior portion of the meristem and is not usually expressed in the SAM or mature AM [wus mutation does not affect axillary meristem development in Arabidopsis, but rice TAB1 seems to control AM formation through mechanisms different from those of WUS in Arabidopsis [TAB1, WOX4 is not expressed in the premeristem but contributes to the maintenance of the AM after almost complete AM establishment. TAB1 forms an AM by enhancing the expression of O. sativa homeobox 1 (OSH1) and WOX4 [OSH1 is preferentially expressed in the AM, and a significant decrease in its expression and a decrease in tillering in its mutant indicate that the gene is necessary for the initiation or maintenance of the fate of undifferentiated cells at the very early stages of AM formation [MONOCULM 3 (MOC3) is a direct homolog of WUS in rice and is necessary for the formation of tillering buds and interacts with key components of the CK pathway controlling rice tillering [Similarly, in rice, itiation . TAB1 isature AM . Interesbidopsis . The othbidopsis . Howeverand WOX4 . During ormation . MONOCULillering . CKs antillering .LAX is expressed on the boundaries of apical meristem- and neomeristem-forming regions, specifically controlling the initiation or maintenance of new meristems [lax1 mutant, and AMs cannot be formed during vegetative development. Similarly, the maize barren stalk1 (ba1) mutant cannot initiate AMs at any stage of the life cycle [MADS34 in rice encodes a MADS-box transcription factor that coregulates the number of primary and secondary branches with LAX1 [MOC1  was the first key transcription factor identified [MOC1 and LAX1 is not altered in the tab 1 mutant, TAB1 plays a role in an independent pathway or downstream of MOC1 and LAX1. In addition, LAX2, together with LAX1 and MOC1, plays a role in different AM maintenance pathways to control branching at vegetative and reproductive stages [LAS in Arabidopsis leads to AM loss during vegetative development, causing loss of branching or tillering and indicating that these genes are highly conserved [LAS and MOC1 are specifically expressed in the initiation region of the AM [moc1 mutant, which lacks axillary buds and tillers, OSH1 expression completely disappears in leaf axils but is not affected in the SAM. Moreover, MOC1 is expressed earlier than LAX1, LAX2 and TAB1 during AM formation, indicating that the sequence or independent role of these genes is the cause of axillary bud formation and that multiple pathways contribute to the development of the AM [LAX PANICLE1 (LAX1) and MONOCULM1 (MOC1) encode bHLH family and GRAS family transcription regulators, respectively, which are necessary for the initiation and maintenance of the AM in rice panicles ,48. LAX eristems . Studiesfe cycle ,50. Thesfe cycle . MADS34 ith LAX1 . MOC1  isolated from sunflower is a homologous gene of ROX/LAX1. In situ results of a cross-section show accumulation of ROXL transcripts at specific points in the boundary region between the apical meristem and the lateral leaf primordium, displaying a similar pattern in Arabidopsis [Ha-ROXL exhibits clear-boundary transcription in vegetative branches, though the expression pattern of LATERAL SUPPRESSOR LIKE (LSL) is confined to the boundary region because signals can also be detected in other cellular domains of vegetative and reproductive branches. Transcription of LSL is also expanded at the early stage of lateral primordium development, indicating that this gene is involved in the early development of the lateral primordium and in the initiation of the AM [Similarly, bidopsis . Based of the AM ,55.BLIND (Bl) is the homologous gene of the RAX1 gene in tomato and Arabidopsis [bl mutant [R2R3 Myb gene regulate axillary meristem formation in a partially redundant manner [ls and bl in tomato display an additive phenotype, suggesting that at least two pathways are involved in AM initiation [FRIZZLE PANICLE (FZP) is a very important gene in rice tillering development. Its overexpression inhibits RFL/PANICLE ORGANIZATION 2 (APO2), which seriously suppresses the formation of axillary meristems at the vegetative stage and leads to a significant decrease in tiller number [BAD1 transcripts are detected mainly in the AM boundary region and between lateral branches, and its functional deficiency results in organ fusion via a reduction in the number and angle of branches [COMPOSITUM 1 (COM1) and RETARDED PALEA1 (REP1), in barley and rice have no similar functions. In addition, BELL1-like homeobox 12 (BLH12) and BLH14 play an important role in maintaining axillary meristems and possess the redundant functions necessary for axillary bud development throughout nutritional and reproductive development [bidopsis ,25. It el mutant . Double-t manner . Double itiation ,22,27. Iitiation . It shouitiation . FRIZZLEr number . In maizbranches . Neverthelopment .Arabidopsis, rice and maize, other species are predicted to have transcription factors involved in branching or tillering. For example, it was found that the formation of the AM in Antirrhinum majus requires ERAMOSA (ERA), a gene encoding a GRAS transcription factor (orthologous to LAS in Arabidopsis). The basic role of ERA in AM formation is consistent with that of LAS/MOC1/ERA in preventing cell differentiation in the boundary area and in stimulating AM formation [In addition to common plants such as ormation .The structure of mature plants is determined by the occurrence of axillary meristems, control of bud growth and subsequent dynamics of branching growth. Changes in these parameters result in the high morphological diversity observed in different plant species and even among individuals of a particular species . After tBRANCHED1 (BRC1) in Arabidopsis is homologous to TEOSINTE BRANCHED 1 (TB1) in maize and plays a core inhibitory role in regulating axillary bud growth , HB40 and HB53 act directly downstream of BRC1 to regulate branching [Arabidopsis, another transcription factor involved in the SL pathway, namely, BRI1-EMS-SUPPRESSOR1 (BES1), negatively regulates cambium activity in the SL signaling pathway. Further studies have shown that SL signaling regulates the expression level of WOX4 through BES1, controlling secondary growth [IPTs, suppressing the growth of axillary buds [FRUITFULL (FUL) in Arabidopsis thaliana, it was found that the combination of auxin and BR strongly induced the growth regulator SMALL AUXIN UPREGULATED RNA 10 (SAUR10) to be directly modulated by FUL, thus participating in branching angle regulation [Arabidopsis branching [Arabidopsis involves a class of photochrome-targeted transcription factors, PHYTOCHROME INTERACTING FACTOR 4 (PIF4)/PIF5 [The transcription factor d growth . BRC2 isd growth . Similarin grape . LAS and of BRC1 . Homeoboranching . Moreoveranching ,62. SL, ranching . In Araby growth . CK is aary buds . In funcgulation . It shouranching . The auxF4)/PIF5 . When thArabidopsis. The gene encodes the AP2/ERF transcription factor and is strongly expressed in proliferating cells. Moreover, overexpression of EBE promotes cell proliferation, shortens the cell cycle in growing calli and stimulates the formation and growth of axillary buds [Overall, the interaction between ABA and auxin may mediate the effect of PIF4/PIF5 on branching . Mohammaary buds .TB1, also known as FINECULM1 (FC1), is a TCP family transcription factor that negatively regulates rice tillering and inhibits the subsequent growth of axillary buds [tb1 mutant exhibits an increase in tiller number [MADS57 is a transcription factor of the MADS domain family that participates in the regulation of axillary bud growth in rice. RNA in situ hybridization analysis has shown that MADS57 is mainly expressed in meristems and axillary buds. Additionally, its expression is higher in the tillering and stem elongation stages than in other stages of rice growth [D14 expression, allowing MADS57 to affect tillering [ary buds . This ger number . In addir number ,71. MADSe growth . InteracCmERF053 was found to be rapidly upregulated in axillary buds when apical dominance was relieved, which may be related to the growth of lateral branches. The gene belongs to the AP2/ERF family and is mainly expressed in stem and root organs. Further transcriptome analysis showed that CK-mediated control of stem branching may be related to the transcription level of CmERF053 [IDEAL PLANT ARCHITECTUREL1 (IPA1), also known as SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 14 (SPL14), is another key regulator that determines plant structure but not meristem activity, and upregulation of IPA1 expression leads to fewer tillers in rice [IPA1 overexpression lines display a reduced tillering phenotype, whereas tillering in the ipa1 mutant increases [D53 is one of the only known transcription targets of SL, and D53 inhibits IPA1 upregulation [D53 promoter and plays a key role in the feedback regulation of SL-induced D53 expression. In summary, IPA1 may constitute a long-term transcription factor that acts with D53 to mediate SL-regulated rice tillering development [D14 transcription to regulate tillering during organogenesis [In a study of branching-related genes in chrysanthemum, CmERF053 . IDEAL P in rice . Furtherncreases . This gegulation . IPA1 dielopment . In lateogenesis .Medicago [SPL13 inhibits axillary bud growth, thereby reducing lateral branches. In this process, MYB112 is targeted and downregulated by SPL13. Compared with WT, MsMYB112 RNAi plants show more branches, which confirms that MYB112 itself also inhibits the lateral branch growth of alfalfa [MdTCP12 [The SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 13 (SPL13) gene encodes an SBP transcription factor that is mainly expressed in meristems and is essential for regulating branching and vegetative growth in Medicago . Overexp alfalfa ,76. Furt[MdTCP12 .Related to ABI3 and Viviparous 1 (RAV1) is a circadian rhythm gene in Castanea mollissima that is homologous to the TEM gene in Arabidopsis. It reaches its peak expression at noon under vegetative growth conditions and is highly expressed during winter dormancy and in response to low temperature [perature . When thFZP and IPA1 in rice, TB1 in maize, and BRC in grape lead to increased branching and increasing yield [Bl in tomato increases the number of branches, also increasing yield. At the same time, the change in plant type caused by branching provides an opportunity to explore the ornamental value of plants. For example, overexpression of CmERF053 significantly increases the number of branches in chrysanthemum. In summary, regulation of these key transcription factors can significantly increase the number of branches, thereby increasing crop yield or quality.Genetic control of branches is the main determinant of yield, seed number regulation and harvestability. For example, interference mutations in ng yield ,48,75,79PIF4 and PIF5 significantly inhibit the branching caused by this shade-avoidance syndrome, providing opportunities for practical cultivation.In addition, fine regulation of branching has become an important strategy for plants to morphologically adapt to various environments. Chestnut CsRAV1, a circadian rhythm response factor, participates in the winter dormancy and low-temperature response of poplar and increases branching. Thus, manipulating this gene may lead to the possibility of producing trees with greater biomass. In actual cultivation, plants usually inhibit axillary bud growth in response to reductions in the ratio of red light to far-red light (R:FR) caused by the presence of competitive neighbors. Overexpression of Triticum aestivum [Rhododendron and Hordeum vulgare [Lilium, FR light strongly inhibits bud outgrowth [2 reduces the negative impact of high temperature on branch growth [It is well known that plant types have remarkable plasticity. Branch development is affected by many external factors, such as light, temperature and soil nutrients. Light is a powerful environmental factor that affects the branching of plants . For exaaestivum , whereasaestivum . Low R:F vulgare . Howeverutgrowth , and bluutgrowth . In geneutgrowth , and stuutgrowth . In summh growth . Of courh growth . Many TFBranching determines plant architecture and crop yield and plays an important role in plant morphogenesis. Therefore, research on branching regulation mechanisms is a popular topic worldwide. The regulation of plant branching by different transcription factors through mutual connections is one of the main directions of the current study of branching development patterns. Previous research and discussion on a single transcription factor have been the premise and foundation for studying the transcription regulatory network.RAV1 in chestnut is a circadian rhythm gene that is homologous to the TEM gene in Arabidopsis. However, the two genes may lead to different phenotypes in woody and herbaceous plants. In view of the transcription factors related to branching discovered to date, we found exploration of the new functions of known transcription factors to be innovative, even though conserved transcription factors appear to provide key targets for the branching regulation mechanism.Notably, the functions of some transcription factors are not conserved. For example,


Numerous studies have highlighted the role of post-translational modifications in the regulation of cell proliferation, differentiation and death. Among these modifications, acetylation modifies the physicochemical properties of proteins and modulates their activity, stability, localization and affinity for partner proteins. Through the deacetylation of a wide variety of functional and structural, nuclear and cytoplasmic proteins, histone deacetylases (HDACs) modulate important cellular processes, including hematopoiesis, during which different HDACs, by controlling gene expression or by regulating non-histone protein functions, act sequentially to provide a fine regulation of the differentiation process both in early hematopoietic stem cells and in more mature progenitors. Considering that HDAC inhibitors represent promising targets in cancer treatment, it is necessary to decipher the role of HDACs during hematopoiesis which could be impacted by these therapies. This review will highlight the main mechanisms by which HDACs control the hematopoietic stem cell fate, particularly in the erythroid lineage. The hypothesis that reversible histone acetylation may control transcriptional activity was proposed in the 1960s . SubsequSince the discovery of the correlation between histone acetylation levels and gene transcriptional activity in 1964 by Allfrey et al. ,30,31. THowever, the relationship between HDACs and gene expression in yeast remains controversial. There is evidence that HDACs are highly present in transcriptionally active genes and may therefore also be involved in transcription ,41. IndeIn addition to histones, several cytoplasmic and nuclear non-histone substrates are also targets of reversible acetylation . PhylogeHighlighting the importance of the acetylation/deacetylation of non-histone substrates in cell physiology, HDAC6, is remarkable because of its predominant cytoplasmic localization, which depends on the nuclear export sequences and the cytoplasmic retention sequence . In the Many studies using pan-HDAC inhibitors have shown that HDACs have a broad spectrum of functions in human and murine hematopoietic stem cells and in more committed cells, particularly in the erythroid lineage. Main functions of HDACs during hematopoiesis are summarized HDAC1 and HDAC2 are class I HDACs that play an essential role in the regulation of HSC homeostasis in conditional knockout murine models. Simultaneous deletion of HDAC1 and HDAC2 results in the loss of HSC and consequently bone marrow failure. The expression level of HDAC1 has been shown to be involved in the cell fate of hematopoietic progenitors. Overexpression of HDAC1 in hematopoietic progenitor cells transplanted into mice favors erythro-megakaryocytic differentiation at the expense of myeloid differentiation. Conversely, HDAC1 knockdown is mediated by small interfering RNA increases in myeloid differentiation and disrupts the erythroid differentiation of progenitor cells. The expression profile of HDAC1 is regulated by hematopoietic transcription factors. Thus, C/EBPs inhibits HDAC1 transcription in normal myeloid differentiation, while GATA-1 activates it during erythroid-megakaryocytic differentiation .HDAC3 is another class I HDAC which was found to negatively regulate human HSC expansion. Specific HDAC3 knockdown significantly enhances CD34+ cell expansion without affecting cell differentiation in vitro . FurtherHDAC8, another member of the class I family, is involved in the maintenance of long-term hematopoietic repopulation. This enzyme interacts with p53 and modulates p53 activity by deacetylation. When exposed to genotoxic and hematopoietic stress, HDAC8-deficient long-term hematopoietic stem cells exhibit increased apoptosis associated with p53 hyperactivation .Class II and class III HDACs are important players in HSC homeostasis and aging. A member of HDAC class IIa, HDAC5, controls HSC homing by downregulating the membrane receptor CXCR4 (chemokine C-X-C receptor-4) transcription though deacetylation of p65, a subunit of NF-κB. The interaction between CXCR4 and SDF-1 is an essential hub of the homing process . Upon inSirtuins, members of the class III family, protect HSCs against aging . SIRT1 i+ cells exposed to IL-3, abrogated it in the presence of EPO. Furthermore, FK228 induces the apoptosis of CD36+GPAlow/− and CD36+GPAhigh erythroid cells under EPO exposure [Experiments conducted both in vivo and in vitro suggest various roles for HAT/HDACs in the erythroid lineage. For example, the HDACi FK228 and TSA-enhanced erythroid cell production from CD34exposure . Since tClass I HDACs are able to interact with specific erythroid lineage-transcription factors, thus playing a role in the regulation of the human γ-globin (huγ) gene. For example, HDAC1 contributes to the Ikaros-mediated repression of huγ during the transition from γ- to β-globin in ontogenic development. Specifically, Ikaros promotes the recruitment of a complex with repressosome activity, which includes HDAC1, GATA1, FOG1, as well as components of the Mi-2/NuRD complex at huγ promoters. The formation of this repressosome decreases the frequency of remote chromatin interactions between the huγ promoters and the locus control region (βLCR). This process thus ultimately leads to specific silencing of the huγ gene . HDAC1 iBy associating with NCOR1 (nuclear receptor co-repressor 1), HDAC3 forms a protein complex which occupies the γ-globin promoter, playing a role in the developmental silencing of fetal globin genes. In contrast, the displacement of the HDAC3/NCOR1 complex mediated by short-chain fatty acids (SFCA) promotes the recruitment of RNA polymerase II associated with an enhancement in histone H3 and H4 acetylation at the huγ gene promoter. Moreover, in human erythroid progenitors, specific siRNA knockdown of endogenous HDAC3 results in an increased transcription at the huγ gene promoter . In addiUnlike HDACs 1 and 3, HDAC9 upregulates γ-globin gene expression. Indeed, chromatin immunoprecipitation (ChIP) assays have shown that HDAC9 binds in vivo in the region upstream of the Gγ-globin gene promoter. Furthermore, small interfering RNA (siRNA) knockdown of HDAC9 in primary human erythroid progenitors results in γ-globin gene silencing. Conversely, the forced expression of HDAC9 simultaneously increases γ-globin and HbF mRNA levels. Because multiple transcription factor binding regions for myocyte enhancer factor 2 (MEF2) have been identified in the γ-globin promoters of the erythroleukemic line K562, it has been suggested that MEF2 may be involved in the recruitment of HDAC9 to these promoters .The KO of HDAC1/2 in hematopoietic cells is lethal in mice, resulting in severe hematopoietic defects predominant in the erythroid lineage, with a high apoptotic rate . HDAC1/2(i) The Sin3A/HDAC1 corepressor complex can interact with EKLF, an essential transcription factor in the activation of the β-globin gene . The CBP(ⅱ) HDAC1 acetylation status also affects the activity of the HDAC1-containing NuRD complex. NuRD is essential for GATA1-mediated gene activation/repression processes. During the early proliferative phase of erythropoiesis, NuRD deacetylates histones via HDAC1 and rearranges chromatin to repress the expression of GATA1 target genes. On the contrary, during GATA1-directed erythroid terminal differentiation, p300/CBP-mediated acetylation inhibits HDAC1 deacetylase activity within the NuRD complex, which then exerts an activating effect on gene expression .(ⅲ) HDAC1 and HDAC2 are also components of a repressor complex that comprises CoREST, the histone demethylase LSD1, and the transcriptional repressors Gfi-1 and Gfi-1b. Gfi-1b, by associating with CoREST-LDS1, recruits these cofactors to the target promoters. Inhibition of CoREST and LSD1 disrupts the differentiation of erythroid, megakaryocytic, and granulocytic cells, as well as primary erythroid progenitors .Another mechanism of the HDAC1-mediated control of erythropoiesis is based directly on GATA1 deacetylation. Indeed, HDAC1 and CBP are required for controlling GATA1 acetylation levels, which regulate its transcriptional activity and erythroid differentiation . In MEL HDAC1 is also involved in the expression of PU.1, a negative regulator of erythroid commitment. Mechanistically, HDAC1 deacetylates TAF9, a member of the TFIID complex, allowing TAF9 to fix the PU.1 gene promoter and activate its expression. During erythropoiesis, acetylation of HDAC1 occurs, which reduces its deacetylase activity and thus promotes acetylation of TAF9. Acetylation of TAF9 disrupts its binding to the PU-1 gene promoter, and disassembles the TFIID complex, resulting in the repression of PUI transcription and the erythroid commitment of multipotent myeloid progenitors ,109,110.Finally, in mice fetal liver cells, HDAC2 knockdown did not affect erythroid cell proliferation, differentiation, or apoptosis, but had a negative effect on nuclear condensation and enucleation, showing that Class I HDACs may have a different level of action on erythroid differentiation, depending on the stages and the nature of erythroid cells .Among the class II HDACs, two enzymes appear to play a particularly important role during erythropoiesis: HDAC5 and HDAC6. HDAC5, a member of HDAC class IIa, plays both a negative and a positive role during erythropoiesis, depending on the cell system and the stage of differentiation. In MEL cells, HDAC5, colocalized with GATA1 and their co expression, repressed the GATA1 transcriptional activity, whereas during MEL differentiation, the complex dissociates and HDAC5 partially shuttles to the cytoplasm . In erytHDAC5 is also involved in the regulation of erythropoiesis through the nuclear remodeling shuttle erythroid (NuRSERY) complex which contains HDAC5, GATA1, EKLF and pERK. This complex allows GATA1 and EKLF transportation from the cytoplasm to the nucleus. ERK phosphorylation is thought to be required for the formation of NuRSERY. Since pERK decreases during erythroid differentiation, the NurSERY complex seems to be predominantly involved in the early steps of erythropoiesis .More recently, Wang et al. showed that HDAC5 expression increased significantly in the later phase of human erythropoiesis and that HDAC5-mediated deacetylation of histone and non-histone proteins was required for the survival, proliferation and enucleation of erythroblasts. Indeed, the lack of HDAC5 resulted in increased apoptosis and impaired enucleation of erythroblasts, and led to the acetylation and activation of the pro-apoptotic molecule p53. Furthermore, in late-stage erythroblasts, HDAC5 deficiency or pharmacological inhibition increased H4 (K12) acetylation, together with decreased chromatin condensation, arguing for an HDAC5 role in chromatin condensation during erythropoiesis .−/− mice expressing mutated JAK2V617F, and the description of myeloproliferative neoplasms associated with LNK mutations in humans [V617F. However, the relevance of HDAC6 control of erythropoiesis via the level of 14-3-3ζ acetylation determining LNK-JAK2 interaction in the context of JAK2V617F mutation remains to be demonstrated. Indeed, recent data have shown that in leukemic cells carrying the JAK2V617F mutation and in murine models of myeloproliferative neoplasms induced by the MPLW515L mutation, HDAC11 more than HDAC6 was required for cell proliferation and survival via the control of the JAK-STAT pathway [HDAC6, belonging to HDAC class IIb, is another deacetylase involved in the enucleation process of terminal erythroid differentiation. Indeed, in mouse fetal erythroblasts, mDia2, a formin family effector protein, is an important player in enucleation because it promotes the establishment of the actomyosin contractile ring (CAR). The acetylation status of lysine, located in the homology 2 domain of formin, is involved in the regulation of mDia. Indeed, its deacetylation by HDAC6 in mouse erythroblasts leads to mDia2 activation, followed by CAR formation at the cleavage furrow and enucleation . Inactivn humans ,128. In n humans . Consequ pathway .HDACs are enzymes capable of deacetylating a wide variety of functional and structural, nuclear and cytoplasmic proteins. They are amongst the promising therapeutic targets in cancer therapy and have led to the development of numerous HDACi. Despite the clinical interest in these molecules, they have side-effects, notably hematological ones, due to the wide range of functions that HDACs exert on hematopoiesis, including the regulation of HSC homeostasis and erythropoiesis. In the erythroid lineage, HDACs are involved at all steps, from the regulation of the erythro-megakaryocytic commitment to enucleation, and from the control of EPO signaling to the globin switch. This remarkable diversity of HDACs function during erythropoiesis occurs through multiple mechanisms, including chromatin accessibility, transcription factor activity or cytoplasmic targets regulating apoptosis, protein quality control pathways or signaling between membrane receptor activation and gene transcription. Thus, it seems crucial to pursue the effort to decipher the role of HDACs during normal, but also pathological erythropoiesis, such as polycythemia vera and myelodysplastic syndrome, keeping in mind that they remain potential selective targets in these diseases.


Epigenetic events, which comprise post-translational modifications of histone tails or DNA methylation, control gene expression by altering chromatin structure without change in the DNA sequence. Histone tails modifications are driven by specific cellular enzymes such as histone methyltransferases or histone acetylases, which play a key role in regulating diverse biological processes. Their alteration may have consequences on growth and tumorigenesis.NSD1 in humans that are the main cause of SOTOS syndrome, a disease associated with an increased risk of developing cancer. We then report the role of NSD1 in triggering tumor suppressive or promoter functions according to the tissue context and we discuss the role of NSD1 in melanoma. Finally, we examine the ongoing efforts to target NSD1 signaling in cancers.Epigenetic regulations, that comprise histone modifications and DNA methylation, are essential to processes as diverse as development and cancer. Among the histone post-translational modifications, lysine methylation represents one of the most important dynamic marks. Here, we focused on methyltransferases of the nuclear binding SET domain 1 (NSD) family, that catalyze the mono- and di-methylation of histone H3 lysine 36. We review the loss of function mutations of  Epigenetic events serve to remodel chromatin structure without nucleotide change, which subsequently lead to gene regulation and expression. They comprise post-translational modifications of histone tails, DNA methylation, nucleosome occupancy, non-coding RNA regulation and RNA editing . Post-trAmong the histone post-translational modifications, lysine methylation represents one of the most important dynamic marks. Histone lysine methylation is deposited, recognized and removed by a set of specific lysine methyltransferases, effector proteins, and demethylases, respectively, whose expression depends on the cellular context . Their dAs such, these modifications play a key role in regulating diverse biological processes such as cell cycle, DNA repair and genomic integrity maintenance amongst a multitude of other processes.NSD1 (nuclear receptor SET (su(var)3–9, enhancer-of-zeste, trithorax) domain containing protein-1/KMT3B) and its two homologs, NSD2 (WHSC1) and NSD3 (WHSC1L1) [In this review, we discuss the role of the Nuclear binding SET Domain 1 (NSD) family of histone H3 lysine 36 methyltransferases in developmental disorders and cancer. This family comprises NSD proteins share a highly conserved catalytic SET domain, further subdivided into pre-SET, SET and Post-SET domains. They exhibit additional domains, such as multiple Proline-Tryptophan-Tryptophan-Proline (PWWP) and Plant Homeodomain (PHD) finger regions, which are responsible for chromatin and protein interactions ,8,9. MulNSD proteins catalyze the mono- and di-methylation of H3K36, which then serve as substrates for trimethylation by SETD2 ,13 FiguA.Methylation of H3K36 is found in species ranging from yeasts to mammals. It plays a crucial role in regulating gene transcription activation, maintaining genomic integrity and stability, DNA repair, cell cycle and even nutrient stress response ,15 FiguA.NSD1 gene, located on chromosome 5q35, encodes two protein isoforms of 2696 aa and 2427 aa. Lucio-Eterovic and coworkers have demonstrated that the short NSD1 isoform is predominantly expressed in a variety of cell types including fetal/adult brain, kidney, skeletal muscle, spleen and the thymus whereas the long isoform is less abundant [NSD1 heterozygous mutant mouse embryo is viable and fertile but NSD1 knockout is lethal, demonstrating its importance in embryonic development [Human abundant ,17. NSD1elopment .NSD1 has the ability to bind near various promoter regions and interact with H3K36 and RNA polymerase II (RNAPII) to regulate transcription and promote gene expression by stimulating the RNAPII-mediated elongation . ThroughNSD1′s haploinsufficiency triggers a specific genome-wide pattern of DNA methylation alterations, which might represent a highly sensitive and robust diagnostic tool for Sotos syndrome compared to other overgrowth development syndromes [NSD1′s disruption affecting H3K36 methylation could, in turn, prevent these histones from recruiting DNA methyltransferases [NSD1′s loss leads to DNA hypomethylation, highlighting a possible interplay between epigenetic histone methylation marks and DNA methylation  Figure B. In linyndromes . It has sferases ,22.NSD1 functions mentioned above contribute to the pathogenesis of a variety of human congenital developmental syndromes and cancerous diseases which shall be discussed further in this review.When altered, the various NSD1 represents one of the key regulators of development. In humans, germline alterations  of NSD1 potentially inducing loss-of-function of the NSD1 protein have been associated with a developmental syndrome called Sotos. Sotos is characterized by a distinctive facial appearance, physical overgrowth, advanced bone age, learning disabilities and cancer predisposition [position ,23,24,25NSD1 gene [NSD1 point mutations and 58 microdeletions have been reported, and the frequencies of each differ among populations. For instance, the Japanese population harbors microdeletions in the majority of cases, whereas in European populations, NSD1 intragenic inactivating mutations account for the majority of Sotos cases [NSD1 alteration and clinical phenotype was reported [NSD1, but there was evidence of clustering of missense mutations in highly conserved functional domains. Likewise, the position of mutations in the patients with Weaver syndrome has been described to reflect a genotype-phenotype correlation [Kurotaki and coworkers established that 77% of individuals diagnosed with the Sotos syndrome are genetically characterized with microdeletions or point mutations impacting the entire SD1 gene . A totalos cases ,27. A streported . In the relation .Moreover, why there are different rearrangement frequencies among the populations studied remain to be determined.NSD1′s alterations have primarily been incriminated in the overgrowth Sotos syndrome, additional NSD1 disruption have been depicted in other developmental syndromes. Indeed, NSD1 mutations have also been observed in unexplained cases of a congenital overgrowth syndrome known as Beckwith-Wiedemann syndrome (BWS), which is characterized by macroglossia, visceromegaly, umbilical hernia, abdominal wall defect and a predisposition to cancer. It is worth noting that microduplications of 5q35.2–q35.3 comprising the NSD1 gene locus have been described in rare patients with a clinically reversed Sotos syndrome. These individuals are characterized by short stature, microcephaly, learning or mild to moderate intellectual disabilities, and distinctive facial features. NSD1 5q35 duplication has been also detected in patients with Silver-Russell syndrome (SRS) features, which is a growth retardation disorder characterized by facial dysmorphia and body asymmetry [Although symmetry . These osymmetry ,30.To understand the mechanisms underlying the Sotos syndrome, Brennan and co-workers studied the transcriptomic and DNA methylation profiles in Sotos syndrome patients and healthy control individuals. Their results indicate that NSD1-deposited H3K36 methylation regulates transcription by directing promoter DNA methylation, partially by repressing polycomb repressive complex 2 (PRC2) activity .NSD1 may result in repression loss of growth promoting genes [Bearing in mind that normally NSD1 binds to both transcription co-factors and methylated histones to regulate gene expression, Kurotaki et al.’s findings suggest that NSD1 could also repress gene expression ,26,30. Hng genes . The mecng genes .NSD1 loss, which translates into reduced H3K36me2 active transcriptional methylation marks, has also been shown to cause a genome wide accumulation of H3K27me3, a repressive histone modification associated with chromatin silencing, mediated by PRC2-catalytic subunit Enhancer of zeste homolog 2 (EZH2) [NSD1 loss-of-function mutations in Sotos syndrome patients was observed [NSD1 mutations from control subjects, as well as from cases with the clinically overlapping Weaver syndrome ensuing mutations in the histone methyltransferase EZH2.2 (EZH2)   ,35. Furtobserved . The sigRecently, Martin-Herranz and coworkers examined the epigenetic clocks, which represent biomarkers for the aging process, in patients with a variety of developmental disorders harboring mutations in proteins of the epigenetic machinery. Using DNA methylation data generated from their blood compared to healthy individuals, the authors demonstrated that loss-of-function mutations in NSD1, which cause Sotos syndrome, substantially accelerate epigenetic aging ,38.Their findings firstly revealed that patients with Sotos syndrome showed strong evidence of accelerated epigenetic aging when compared with healthy individuals, which makes their epigenome look a decade older than expected . SecondlAPC2), a tumor suppressor gene involved in the negative regulation of the Wnt/β-catenin signaling pathway [One of NSD1′s potential downstream effectors in Sotos syndrome is Adenomatous polyposis coli 2  repression of genes normally active such as tumor suppressor genes, (2) activation of genes normally silenced such as oncogenes. The emergence of these mechanisms is conducted either by a single or a team effort of a variety of histone modifying enzymes . Among tNSD1 expression varies across tumor types  genes, including several collagen and laminin genes, were found in NSD1-mutant tumors [NSD1-mutant tumors may favor immune recognition and tumor elimination [Nevertheless, HPV-negative HNSCC with NSD1 mutations display better treatment responses to platinum-based chemotherapy compared to those lacking these mutations . Of noteA levels , and inaA levels ,61. Furtpression . Anotherrognosis . The tott tumors . Since tmination .SETD2 somatic mutations as well as high EZH2 expression [NSD1′s hypermethylation level was observed in metastatic kidney tumors and often predicted advanced cancer stage and poor overall patient survival [Furthermore, Su et al. showed in vitro that ccRCC samples harbored a pattern of DNA hypermethylation in NSD1′s promoter regions which correlated with NSD1′s silencing and with pression . High NSsurvival . Moreovesurvival .SETD2 have been also reported in ccRCC, and this might occur through a possible crosstalk between the inactivation of NSD1 by methylation and SETD2 [Alterations of nd SETD2 .NSD1 displayed a specific genome-wide DNA methylation pattern consistent with the methylome signature observed in Sotos syndrome and HNSCC [Supporting the role of NSD1 loss in ccRCC tumorigenesis, Yan and colleagues found in RCC samples (TCGA cohort) a much higher level of NSD1 amplification in over 10% of ccRCC samples which was associated with a significant prolonged survival . The mecnd HNSCC ,55,63. Tnd HNSCC .Moreover, in altered H3K36 methylation settings, H3K27me3 would accumulate in regulatory elements of tumor suppressor genes due to EZH2-function, leading to their transcriptional repression, subsequently initiating poor cell differentiation, vascular invasion and tumor progression.NSD1, through promoter hypermethylation, has been associated with neuroblastoma and gliomas. Moreover, in breast tumors, NSD1 gene silencing was associated with reduced sensitivity to tamoxifen [Epigenetic silencing of amoxifen , therebyNSD1 has been implicated in multiple tumor types, particularly hematological malignancies. The first mention of NSD1 role in tumorigenesis comes from the discovery in childhood acute myeloid leukemia (AML) of a translocation at t   encoding a chimeric protein encompassing the carboxyl terminal of NSD1 that retains among other the catalytic SET domain fused to the FG-repeat domain of the nucleoporin protein NUP98, a component of the nuclear core complex. The NUP98-NSD1 fusion protein, reported in 5% of the cases, allows both H3K36 methyltransferase activity through NSD1 SET domain, and histone acetyltransferase activity via the NUP98 domain FG-repeats that interact with the histone acetyltransferase CBP/p300 [Hox-A and Meis 1, which subsequently leads to their transcriptional activation and contribute to leukemogenesis [Hox-A locus activation and AML induction. The epigenetic mechanism behind this activation could be insured by H3K36me blocking EZH2 complexes from binding to regulatory elements, hence preventing H3K27me3 mediated chromatin silencing.Somatic disruption of CBP/p300 ,68. LittRecently, Schmoellerl et al. identified CDK6 as a direct target of NUP98-NSD1 fusion protein in AML. CDK6 was found to be highly expressed in a novel mouse model allowing for regulatable expression of the NUP98-NSD1 signaling axis. CDK6 inhibition via a genetic or pharmacological approach using palbociclib led to a decreased leukemogenesis, with a rapid induction of apoptosis and cell cycle arrest .Translocations involving NSD1 have been also detected in breast cancer .In human HCC, NSD1 expression is higher in tumor tissues relative to normal tissue and its expression is associated with a reduced overall survival . The autIn the same vein, in human colon cancer cell line HCT116, NSD1 has been reported to bind to the promoter proximal regions of a panel of genes, some, such as bone morphogenetic protein 4, may mediate its role in cancer and Sotos syndrome. Mechanistically, NSD1 may interact with H3K36 and RNA polymerase II (RNAPII) to regulate transcription and promote gene expression by stimulating the RNAPII’s elongation process [In laryngeal tumors, characterized by high recurrence and poor overall survival, NSD1/2 mutations are associated with better prognosis, which provide relevant molecular prognostic biomarkers for treatment stratification .While NSD1 has a selective nucleosomal preference for histone 3, it has been demonstrated that NSD1 has a non-histone substrate selectivity as well. Indeed, NSD1 has the ability to activate the p65 subunit of NF-KB, which is a central coordinator for immune and inflammatory responses, via the methylation of lysine 218 and 221 .In conclusion, depending on the cellular context, and the chromatin modifying alterations, NSD1 may function then, as either an oncogene or as a tumor suppressor . This coCutaneous malignant melanoma is the deadliest form of skin cancer that originates from melanocytic cells. Although major breakthroughs were realized in the treatment of advanced melanomas these last decades, the responses are either transient or limited to restricted subsets of patients due to intrinsic or acquired resistance . SeveralNSD1 alterations in 12% of the cases (n = 43/363). The highest frequency of alterations was attributed to missense mutations (n = 32), most of them occurring prior to the NSD1 SET catalytic domain. The other alterations included amplifications (n = 3), truncating mutations (n = 4), homozygous gene deletion (n = 1) (deep deletion) (Skin cutaneous melanoma (SKCM TCGA dataset) showed eletion) B.NSD1, specifically the ones located on the SET domain, might result in an inactive truncated NSD1 protein unable to perform its methyltransferase activity, which might create an epigenetic regulatory imbalance. Cutaneous melanoma has a very high prevalence of NSD1 somatic mutations comparable to other cancer types that were discussed previously in this review   C. NSD1 atal.org) B.APC caught our intention given the implication of APC2-related member in Sotos syndrome  and its APC’s promoter region; therefore the expression of the tumor suppressor APC remains repressed, subsequently the Wnt/β-catenin pathway will be constantly active, which would favor melanoma progression.By analogy with Sotos syndrome, we propose that in malignant melanomas, APC operates downstream NSD1. When NSD1′s activity is altered, the H3K36 active mark will not accumulate on 2 seems to be highly involved. EZH2′s high expression is associated with aggressive tumor subgroups [In the pathogenesis of melanoma, another epigenetic component known as EZHubgroups ,88, and ubgroups ,89.EZH2′s repressive epigenetic mark H3K27me3 is highly expressed in metastatic melanomas in comparison to primary melanomas . The samNSD1′s loss-of-function is associated with a reduced epigenetic activating mark H3K36me1/2, therefore creating an epigenetic imbalance in favor with the accumulation of H3K27me3 on promoter regions of APC, leading to Wnt/β-catenin pathway regulation and subsequently to melanoma progression and immune resistance , PD-L1 (Programmed cell Death protein ligand 1) and CTLA-4 (Cytotoxic T-Lymphocyte-associated antigen 4) can become resistant and harbored an upregulated EZH2.The clinical use of EZH2 inhibitor, GSK503, controlled melanoma growth, restored tumor immunogenicity among these patients, and reversed resistance to tumor immunotherapy . Hence, Another selective small molecule inhibitor of EZH2 methyltransferase activity, GSK126 acts on H3K27me3 global reduction and reactivates critical target genes that were previously repressed . A reducMore recently, a selective small molecule inhibitor of EZH2 “Tazemetostat (EPZ-6438)” has been clinically tested in patients with relapsed or refractory solid tumors, non-Hodgkin’s lymphoma, or histiocytic disorders with EZH2 and other gene mutations. The preliminary results from the phase II clinical trial demonstrated that 90% of patients with follicular lymphoma harboring an EZH2 mutation showed an objective response. This compound is on the verge of FDA approval following promising clinical trial results .Recently, NSD1 loss has been reported to cause resistance to EZH2 inhibition  indicatiIt is worth noting that apart from EZH2, a variety of downstream effectors of NSD1 has been reported in different cancers . Inhibita methyltransferase that controls H3K36 methylation, an epigenetic mark usually associated with transcriptional activation. In humans, germline alterations of NSD1 potentially inducing loss-of-function of the NSD1 protein have been associated with a developmental syndrome called Sotos. Sotos syndrome is characterized by overgrowth in early childhood and cancer predisposition. How NSD1 is regulated and functions in cancers is largely unknown.NSD1 is NSD1 has been associated with tumorigenesis. Depending on the cellular context, NSD1 exerts tumor suppressive or promoter effects. Further studies are needed to get insight into the molecular mechanism underlying its effects. Thus, in specific settings targeting NSD1 or its downstream effectors may be a potential strategy for tumor therapy.Somatic dysregulation of


Cancer immunotherapies are highly potent and are gaining wide clinical usage. However, severe side effects require focusing effector immune cell activities on the tumor microenvironment (TME). We recently developed a chimeric antigen receptor tumor-induced vector (CARTIV), a synthetic promoter activated by TME factors. To improve CARTIV functions including background, activation levels, and synergism, we screened a library of promoters with variations in key positions. Here, we present a screening method involving turning ON/OFF stimulating TNFα and IFNγ cytokines, followed by sequential cell sorting. Sequencing of enriched promoters identified seventeen candidates, which were cloned and whose activities were then validated, leading to the identification of two CARTIVs with lower background and higher induction. We further combined a third hypoxia element with the two-factor CARTIV, demonstrating additional modular improvement. Our study presents a method of fine-tuning synthetic promoters for desired immunotherapy needs. Spatiotemporal control over gene expression may improve engineered immune cells.Immune cells are programmed to express effector genes when needed. Interestingly, few genes are transiently induced following activation by external ques2. Effector immune cells that recognize and attack tumor antigens have already gained clinical approval for chimeric antigen receptor T-cells (CAR-T)3. However, the vigorous activities of CAR-T are also a risk factor4. CAR-T responds to even minute amounts of antigens and may cause severe cytokine storm syndrome, requiring additional regulation of the engineered cells5. To reduce life-threatening side effects, CAR-Ts are being developed with an intrinsic self-destruction system6, or with ON/OFF switches8. Improving the spatiotemporal regulation of CAR-T and other types of engineered immune effectors will create safer applications of such robust tumor-killing cells9.Immune therapies can eliminate cancerous cells within the body10. CARTIVs are built by combining binding sites that respond to factors present within the TME. Since tumor microenvironments diverge from normal tissues11, we thought to utilize TME factors as inducers for effector immune cells to act against cancerous cells, and spare normal tissues10. This is somewhat recapitulating the endogenous immune system control of inducing activity within inflammatory sites in vivo, thus reducing collateral damage to healthy tissues. Our initial CARTIV promoters demonstrated specific responses to some of the major inflammatory cytokines, such as gamma-interferon (IFNγ) and tumor necrosis factor-alpha (TNFα), together with hypoxia. Importantly, numerous studies have attempted to develop such tumor-specific promoters, taking different approaches15. We developed the CARTIV approach by combining several minimal binding sites and defined spacers, which resulted in nontrivial findings regarding the number of sites and their relative positions in the promoter10. Our CARTIV promoters are rather short, ranging between 200 and 300 bases only, and modular, to allow further adjustments and specificity to TME factors. Notably, the first study used basic elements that are not necessarily the best binding sites for background expression or maximal fold induction, creating interest in further improvements.We recently published a novel approach to regulate effector immune cells using promoters that are inducible by the tumor microenvironment (TME)16. Even with advanced protein structure prediction17, it is not easy to predict the complex interactions of several factors with DNA and their cumulative induced transcription. Natural promoters have evolved to provide adequate induction of genes, with divergent response time and magnitude. Changes in the sequences of CARTIV promoters will lead to changes in responses to given stimuli. Specifically, changing only several defined key positions within a given promoter may result in a stronger or more specific response, which is of great interest for clinical applications. Optimization of sequences through screening of libraries is well established20, with a focus on key positions being possible21. For example, a random DNA sequence of 12 bases has a complexity of 412 (over 16 million sequences); rational focus on key positions within binding sites may allow manageable permutations for even a 100-base-long promoter.Promoters have "canonical" binding sites of transcription factors, allowing the synthetic design of artificial sequences that may have specific gene expression potency23. While chromosomal positional effects may have an impact on gene expression; we transduce lentiviruses as batch and study the population of cells, not clones. Importantly, several recent publications scanned promoter libraries with lentivirus, successfully25. Hence, we choose to use the same vector as our recent publication10.We choose lentivirus as a transfection vector since it is the accepted practice of CAR delivery into T cells, both in research and in the clinic10 are based on canonical binding sites. IFNγ signaling through STAT1 includes binding with IFNγ activation site GAS, consensus sequence TTCCNGGAA26. TNFα signaling through NF-kB binds with p50, consensus sequence GGRRRTTYYC, where R is A or G, and Y is C or T27. The hypoxia response elements may include HIF consensus sequence RCGTG28. We simplified these core sequences and added rationally designed repeats and spacers10. Notably, we already achieved nontrivial results regarding the order by which basic elements were assembled when combining hypoxia plus GAS plus kB elements10. Improving CARTIV, or any inducible promoter requires screening of focused libraries by functional selection for the desired phenotype. CARTIV promoters already show a good response to TME, but may benefit from additional improvements for specific expression levels.The original CARTIV promoters5 complexity, transduced cells in culture, and screened them through subsequent rounds of sorting with and without activating cytokine. Sequencing identified multiple enriched variants of interest. We further validated some 15 candidates and found improved properties over the original CARTIV promoters. Importantly, we noted that exceptionally high induction or very low background may require different promoter sequences. We also demonstrated that variable nucleotides may have both direct and indirect interactions with their cognate transcription factors. Taken together, we present a method for the functional screening of hundreds of thousands of variant promoters and the identification of improved functionalities.In this study, we aimed to improve CARTIV promoters by functional screening of variations on a theme. Improved promoters may provide nontrivial properties such as robust induction, low background, or synergism. First, we focused on critical nucleotides predicted to allow for variations without losing transcription-factor binding. We constructed libraries limited to less than 10The library sequence used:cccggtcgcactagttctagaAYTTCCSGGAARTAGGGTGGGCAAGTAYTTCCSGGAARTtctagaGGRRRTTYYCGGGGACTTTCCGGRRRTTYYCtctagaTATTAAGGTGACGCGTGTGGCCTCGAACACCGAGCGACCCTGCAGCGACCCGCTTAAAAgcggccgccATG.The oligo, 4 nmol, was ordered from Integrated DNA Technologies . The library was amplified (15 cycles) using PrimeStar© max  with primers containing SpeI and MluI restriction enzyme sites. DNA fragments were resolved on agarose gels, extracted, digested, and cloned into pHAGE2 plasmids containing fluorescent reporters. Plasmids were electroporated into NEB 10-beta cells that were grown overnight in 300 ml LB and then extracted using a midiprep kit .l-glutamine, non-essential amino acids, and sodium pyruvate . Cells were grown at 37 °C in a humidified 5% CO2 incubator. Human recombinant IFNγ and TNFα with activities of 2 × 107 Units/mg were purchased from PeproTech . Standard lentivirus production was as reported10.HEK293T cells were grown in DMEM containing 10% serum, Pen-Strep, HEPES, 10. Briefly, human PBMCs were isolated from a healthy consenting donor’s blood by Ficoll gradient. Cells were activated using soluble anti human CD3 OKT3  for 48 h and transduced using RetroNectin  coated plates according to the manufacturer recommendations. Two days following transduction culture was stained for CD3 to validate T cell expansion.Primary T cell culture were established, maintained and transduced as described elsewhere5 cells per well in 96-well flat-bottomed plates. Cytokines were added to final concentrations of 500 U/ml. In experiments involving hypoxia, cells were cultured in a hypoxic chamber with a gas mixture of 5% CO2, 0.3% O2 and 94.7% N2 at 20 l/min for 3–5 min, and then sealed and placed at 37 °C for 16–20 h before analysis. Cells were harvested, suspended with DAPI 1 µg/ml and measured by FACS using a Beckman Coulter® Gallios™, Cytoflex or CytoFlexS flow cytometers. Data were analyzed using Kaluza™ or the CytExpert software.HEK293T cells were plated at 1 × 106/10 cm plate. Cells were transduced the following day at an MOI of < 0.3 . Cells were harvested and re-plated for activation at least 72 h post-transduction. Cells were sorted by fluorescent-reporter expression using a FACS aria III (BD) cell sorter for each of the three rounds of selection.HEK293T cells were plated at 3 × 10Genomic DNA was extracted and promoters were amplified by PCR for 22 cycles and sent for next-generation sequencing (NGS) at HyLabs . The average number of sequences per sample was 22,368. BioSample metadata are available at the NCBI BioSample database under accession SAMN 21397074: CARTIV G1K1 Library (TaxID: 9606)., where ‘x’ is the normalized GeoMean, ‘G’ and ‘K’ are IFNγ and TNFα treatments, respectively, and ‘bg’ is background GeoMean. When no G or K present, the GeoMean is of cells expressing miniTK alone.Potency was calculated using the equation FACS data are shown as mean ± SD. Data are representative of at least three independent experiments unless otherwise noted. A two-tailed T-test was performed, with p < 0.05 considered significant.10, but were not optimized. In order to improve functional parameters of CARTIV promoters, such as reduced background, increased induction, and synergism, we designed a library based on CARTIV promoter G1K0.6 . The primary design and the 3′-positions were selected based on the IFNγ-PRE consensus30. We introduced a tandem repeat of this core into the template, separated by a linker AGGGTGGGCAAGT ). By this analysis, the combination of cytokines had the most synergistic effect in sequences 5 and 130 . FACS analysis measured induction of the fluorescent reporter Fig. b. SequenAn important parameter for CARTIV promoters is their background level, i.e., expression without cytokines. To determine background, we measured the fluorescence levels of cells transduced with vectors having the miniTK promoter only, and compared them with the selected CARTIVs without cytokines. Data indicated that sequences 90 and 130 had the lowest background . In contrast, sequences 11, 79, and 143 had the highest background . In addition, other variants showed low background, among them sequence 5  and tracked using a lionheart fx automated microscope. Seq 5 showed a faster ON rate  and HG1K06-5 (H5) were cloned and transduced into fresh cells. After expansion, the cells were tested without cytokines, with IFNγ, with TNFα, or with both, and subjected to hypoxic conditions for the last 18 h before reading signal intensity. In agreement with previous experiments, the combination of IFNγ and TNFα resulted in high induction. The hypoxia HCPRE further enhanced the reporter levels  by adding it to CARTIV promoters10. Notably, both 130 and H130 had higher maximum induction compared to the original G1K06 and G1K06H1 respectively  identified numerous candidates that were validated independently, revealing nontrivial combinatorial effects of key nucleotides. We demonstrate improvements including low background and high fold activation in response to two factors characteristic of TME. Moreover, we show that these improved promoters can further benefit from the addition of a third hypoxia element, demonstrating the modularity of the CARTIV design.10; however, activation by IFNγ alone was relatively low. The basic GAS PRE element has the typical sequence for STAT binding26. Variations of selected nucleotides . For library construction we used AYTTCCSGGAARTG . However, this may cost substantial background expression and reduction in synergismism Fig. . The metism Fig. . Such imUsing lentiviruses for screening of promoter libraries is non-trivial. While we cannot rule out positional effects, the fact that we see reproducible changes of activity between promoters with only a few mutations, in independent experiments having independent batch transductions, strongly suggests that our CARTIV promoters are the main effector on reporter expression.. showed that a single SNP in a DNA binding recognition site can influence transcription factor binding, thereby affecting gene regulation39. Thus, our engineering of synthetic promoters is also relevant for natural variations among humans. One may assume low or no tolerance for variability where a nitrogenous base interacts directly with a transcription factor. Interestingly, Le et al. demonstrated that dinucleotides flanking the core promoter sequence can contribute significantly to transcription factor binding40, thus adding fine-tuning of activities. Nevertheless, as we have demonstrated, a wide range of nontrivial properties can be observed when screening the variable positions of a known consensus sequence, with additional complexity when using multiple binding sites.According to structural data, the selected variable positions are within the DNA–protein interactions Fig. . The hig10, thanks to the abundance of inflammatory cytokines such as TNFα and IFNγ (36). TNFα and IFNγ might also be present in inflammatory sites other than TME. Therefore, additional PREs that correspond to TME and not to inflammatory sites will help41. Hypoxia is a hallmark of TME42, and we demonstrated the possible addition of an HCPRE to CARTIV10. The cumulative improvement of HCPRE with Seq 5 or Seq 130 (Fig. Our library was based on our basic CARTIV promoter designed to regulate effector gene expression within the TMEnd IFNγ 3. TNFα an43. CARTIV promoters with variations and modular modifications can provide engineered immune cells for specific TMEs.We present an approach to designing, screening, and functionally validating inducible promoters with different traits that may improve engineered immune cells. Yet, in order to evaluate the efficacy of treatments employing these promoters they should be coupled to an appropriate CAR and evaluated in an in-vivo model. The dogma of “one treatment fits all” is shifting towards precision treatmentSupplementary Figures.Supplementary Table 1.Supplementary Table 2.


The internal promoter in L1 5’UTR is critical for autonomous L1 transcription and initiating retrotransposition. Unlike the human genome, which features one contemporarily active subfamily, four subfamilies  have been amplifying in the mouse genome in the last one million years. Moreover, mouse L1 5’UTRs are organized into tandem repeats called monomers, which are separated from ORF1 by a tether domain. In this study, we aim to compare promoter activities across young mouse L1 subfamilies and investigate the contribution of individual monomers and the tether sequence.We observed an inverse relationship between subfamily age and the average number of monomers among evolutionarily young mouse L1 subfamilies. The youngest subgroup (A_I and Tf_I/II) on average carry 3–4 monomers in the 5’UTR. Using a single-vector dual-luciferase reporter assay, we compared promoter activities across six L1 subfamilies  and established their antisense promoter activities in a mouse embryonic fibroblast cell line and a mouse embryonal carcinoma cell line. Using consensus promoter sequences for three subfamilies , we dissected the differential roles of individual monomers and the tether domain in L1 promoter activity. We validated that, across multiple subfamilies, the second monomer consistently enhances the overall promoter activity. For individual promoter components, monomer 2 is consistently more active than the corresponding monomer 1 and/or the tether for each subfamily. Importantly, we revealed intricate interactions between monomer 2, monomer 1 and tether domains in a subfamily-specific manner. Furthermore, using three-monomer 5’UTRs, we established a complex nonlinear relationship between the length of the outmost monomer and the overall promoter activity.The laboratory mouse is an important mammalian model system for human diseases as well as L1 biology. Our study extends previous findings and represents an important step toward a better understanding of the molecular mechanism controlling mouse L1 transcription as well as L1’s impact on development and disease.The online version contains supplementary material available at 10.1186/s13100-022-00269-z. Long interspersed elements type 1  are ubiquitous non-long terminal repeat (LTR) retrotransposons in mammals , comprisGenomic L1 sequences are grouped into subfamilies according to their evolutionary history. Among L1s in the human genome, the oldest subfamilies L1MA to L1ME are shared with other mammals, but the younger L1PB and L1PA subfamilies are only found in primates. The youngest subfamily, L1PA1 , is specific to humans . A remarThe laboratory mouse is an important mammalian model system for human diseases as well as L1 biology –33. Despspa, a Tf subfamily member [Owing to their lineage-specific nature human and mouse L1 5’UTRs share no sequence homology. Moreover, mouse L1 5’UTRs are distinctly different from human L1’s in that the former are organized into tandem repeats called monomers , 36. Sucy member . Similary member . In bothy member , 41. In R = -0.91, p = 0.004).To profile mouse L1 promoter activities, we first analyzed the length distribution of mouse L1 5’UTRs by counting the number of monomers for full- or near full-length elements. Since elements from the old subfamilies would have accumulated numerous debilitating mutations, we limited our analysis to seven recently active subfamilies, including A_I, Tf_I, Tf_II, Gf_I, Tf_III, A_II, and A_III (listed from young to old). The estimated age for these L1 subfamilies ranges from 0.21 MYA for A_I to 2.15 MYA for A_III  is 914, which can be interpreted as that human L1RP 5’UTR possesses a promoter activity 914-fold above the assay background. As pCH117 usually shows the highest promoter activity among all the constructs tested, its normalized promoter activity is also an indication of the assay dynamic range. Note the assay dynamic range fluctuates to some extent from experiment to experiment , likely due to unpredictable variations in cell status and transfection procedures. However, such fluctuations should not substantially alter the relative fold difference among promoters.To compare promoter activities across mouse L1 subfamilies, we first synthesized the consensus 5'UTR sequence of six subfamilies . As the length of the consensus 5’UTR varies among these subfamilies , we retaactivity . Second,R = -0.62, p = 0.19)  , followed by A_I 274-fold), Tf_I 214-fold), Tf_III (189-fold), A_II (114-fold), and the lowest activity in the Gf_I subfamily (59-fold) . Thus, we consider L1spa as a member of the Tf_I subfamily.DeBerardinis and colleagues have previously investigated the interactions among monomers and the tether sequence based on a single promoter variant, L1 element , 39. Spespa 5’UTR [p < 0.05 via pairwise t-test with Benjamini–Hochberg correction for multiple testing; adjusted p values for all pairwise t-tests are provided in Additional file p < 0.05). To confirm such residual promoter activities, we included two additional control plasmids . Second, we placed the tether sequence in its antisense orientation (T_AS). Interestingly, the antisense Tf_I tether had 8.2-fold higher activity than the assay background (p < 0.05). These results suggest that the Tf_I tether sequence has some weak transcriptional activities in both sense and antisense orientations. To aid in the interpretation of the contribution of individual domains, we diagrammed promoter activities along with domain locations in an integrated manner . However, combining M1 and T together did not lead to any substantial increase in promoter activity (tenfold above background for M1-T). The deletion of M1 from M2-M1-T reduced the promoter activity by a mere 7% , suggesting M1 contributes little to the overall two-monomer promoter. On the other hand, the presence of tether sequence reduced M2 activity by fourfold , indicating that A_I tether significantly suppresses the promoter activity of M2 and likely plays a negative role in the context of two-monomer promoter. Thus, M2 dominates in its contribution to the overall A_I promoter activity. Similar to the experiment with Tf_I promoters, a 202-bp fragment from the GFP coding sequence (GFP202), equivalent to the length of A_I tether, showed little promoter activity . The antisense A_I tether had threefold higher activity than the assay background (p < 0.05). These results suggest that the A_I tether sequence also has some weak transcriptional activities in both sense and antisense orientations. To summarize, M2 is the major contributor of two-monomer promoter activity for A_I subfamily, the tether negatively regulates M2 activity in the context of two-monomer 5'UTR, while the role of M1 is minimal , although the magnitude of reduction was not as dramatic as in A_I and Tf_I. The activities of individual domains, M2, M1 and the 313-bp tether, were 20.2%, 9.8%, and 20.4% of M2-M1-T, respectively, but remain significantly above the assay background (p < 0.05). The antisense 313-bp tether (T_AS) also had substantial amount of promoter activity . Note the 313-bp tether includes a truncated 64-bp monomer at its 5’ end. We also subcloned the tether sequence without the 64-bp truncated monomer. The shortened 249-bp tether had detectable activities in both sense  and antisense orientation . The interactions among individual domains for subfamily Gf_I are distinctly different from both Tf_I and A_I , suggesting M1 plays a minor role in Gf_I subfamily. Thus, the two-monomer activity of Gf_I is mainly the result of interaction between M2 and tether. Comparable results were obtained from F9 cells despite the overall weaker activity of Gf_I promoter sequences in F9 cells . For Tf_I subfamily, the three-monomer promoter was 2.4-fold higher than the two-monomer version and was only 17.4% lower than the reference L1RP promoter  . Deletion of the first 120 bp had additional effect (down to 23.6% of the three-monomer construct) (p < 0.05 between 80-bp and 120-bp deletion constructs). However, this diminishing trend was reversed when the promoter was further truncated. The promoter activity was restored to 31.6% of the three-monomer construct when the first 160 bp was deleted . The deletion of the entire third monomer (212 bp), giving rise to the two-monomer construct, restored the activity to 42.3% of the three-monomer construct (p < 0.05 between 160-bp deletion construct and M2-M1-T construct). Similar patterns were seen with the vector series for A_I subfamily  Fig. A and 26.RP 5’UTR showed 106.2-fold activity above the assay background, equivalent to 11.6% of that of the sense promoter. In this context, all six L1 subfamilies demonstrated detectable levels of antisense promoter activities (p < 0.05 as compared pairwise to assay background)  far exceeds that of Tf_I (7.7% of L1RP) and Gf_I (1.2% of L1RP) in NIH/3T3 cells . The relative contribution of M2, M1, and tether domains to the overall antisense promoter activity remains unclear. When the tether sequence from subfamily Tf_I, A_I, and Gf_I was tested in the antisense orientation in NIH/3T3 cells, it showed 2.9%, 1%, and 26.5% of the corresponding two-monomer promoter, respectively  and two cryptic spice acceptor sites  [This study has several limitations. The first is that all promoter activities were measured by a luciferase-based reporter system. Although widely adopted, a reporter system that is based on protein activity may be confounded by uncharacterized post-transcriptional regulation, including alternative splicing and polyadenylation . The proctively) . Alternactively) . All thectively) , some asctively) . Thus, actively) ). Lastlyctively) , 59, theThe multimeric nature of mouse L1 5’UTRs presents a challenge to investigate mouse L1 transcriptional regulation. Accordingly, unlike the human L1 5’UTR, many aspects of mouse L1 transcription remain poorly understood. In this study, aided by synthetic biology and report assays with a wide dynamic range, we compared sense promoter activities and discovered antisense promoter activities from six evolutionarily young mouse L1 subfamilies. Expanding upon a pioneering study featuring a single Tf_I element, we determined contribution of monomer and tether sequences among three main lineages of evolutionarily young mouse L1s: A_I, Tf_I and Gf_I. Our work validated that, across multiple subfamilies, having the second monomer is always much more active than the corresponding one-monomer construct. For individual promoter components , M2 is consistently more active than the corresponding M1 and/or the tether for each subfamily. More importantly, we revealed intricate interactions between M2, M1 and tether domains and such interactions are subfamily specific. Using three-monomer 5’UTRs as a model, we established a complex nonlinear relationship between the length of the outmost monomer and the overall promoter activity. Overall, our work represents an important step toward elucidating the molecular mechanism of mouse L1 transcriptional regulation and L1’s impact on development and disease.BLAST + , a suite of command-line tools to run BLAST locally , was useA detailed list of the promoter constructs, including primers and the corresponding promoter sequences, is provided as supplemental tables  or Twist Biosciences. Primers were designed to serially truncating M3 by 40-, 80-, 120-, and 160-bp from the 5’ end. The resulting PCR products were SfiI digested and ligated into SfiI-digested pCH036, giving rise to pLK094, pLK095, pLK096, and pLK097. The two-monomer Tf_I promoter in pLK050 was derived from a synthetic DNA fragment. Primers were designed to amplify M2, M1, and T. The resulting PCR products were digested and ligated into pCH036, resulting in pLK057, pLK056, and pLK054. The antisense version of the tether fragment was similarly cloned into pLK055. M2-T sequence in pLK098 and M1-T sequence in pLK047 were derived from synthetic DNA fragments.The three-monomer A_I consensus promoter in pLK085 was derived from a synthetic DNA fragment. Primers were designed to serially truncating M3 by 40-, 80-, 122-, and 160-bp from the 5’ end. The resulting PCR products were SfiI digested and ligated into SfiI-digested pCH036, giving rise to pLK090, pLK091, pLK092, and pLK093. The two-monomer A_I promoter in pLK049 was derived from a synthetic DNA fragment. Primers were designed to amplify M2, M1, M1-T and T. The resulting PCR products were digested and ligated into pCH036, resulting in pLK053, pLK052, pLK040 and pLK041. The antisense version of the tether fragment was similarly cloned into pLK042. M2-T sequence in pLK046 was derived from a synthetic DNA fragment.The two-monomer G_I consensus promoter in pLK051, the M2-T promoter in pLK099, the M1-T promoter in pLK048 were derived from separate synthetic DNA fragments. Primers were designed to amplify M2 and M1, respectively. The resulting PCR products were digested and ligated into pCH036, resulting in pLK063 and pLK062. Two different lengths of tether were considered. Primers were designed to amplify and clone the tether as a 313 bp fragment in either sense (pLK060) or antisense orientation (pLK061). A shortened 249 bp version of the tether was also cloned in either sense (pLK058) or antisense (pLK059) orientations.RP promoter in pCH117, respectively. To make these antisense promoter constructs, primers were designed to amplify the sense-oriented promoters from the respective precursor constructs so resulting PCR fragments would reverse the orientation of the promoter with respect to the two heterotypic SfiI sites.The two-monomer consensus promoters for A_II (pLK087), Tf_II (pLK088), and Tf_III (pLK089) were derived from separate synthetic DNA fragments. pJT01, pJT02, pJT03, pJT04, pJT05, pJT06, and pJT07 contain antisense versions of the two-monomer promoters in pLK049, pLK050, pLK051, pLK087, pLK088, pLK089 and of the L1We maintained a subline of NIH/3T3 mouse embryonic fibroblast cells in our lab. To confirm cell identity, we submitted an aliquot of the cells to American Type Culture Collection (ATCC) for mouse short tandem repeat (STR) testing. The testing involved the analysis of 18 mouse STR loci as well as two specific markers to screen for potential cell line contamination by human or African green monkey species . The STR2 incubator (48 h for NIH/3T3 cells or 24 h for F9 cells). To measure promoter activity, cells were processed using Promega’s Dual-Luciferase Reporter Assay System. To minimize assay background, all steps were conducted in dark. Firefly luciferase and Renilla luciferase signals were sequentially measured on a GloMax Multi Detection System (Promega). Signal integration time was set to one second per well. Mock transfected cells and empty wells were included to evaluate the assay background.Assays were performed in 96-well format. NIH/3T3 cells were first trypsinized from a stock dish, diluted into a suspension at 200,000 cells per ml in complete medium, and kept at 37 °C before seeding into a 96-well plate. F9 cells were grown in a stock dish coated with 0.1% gelatin, trypsinized, and diluted into a suspension at 400,000 cells per ml before seeding into a 96-well plate coated with 0.1% gelatin. Lipofectamine 3000 (Invitrogen) was used following a reverse transfection protocol. Briefly, for each plasmid, two separate tubes were prepared. In one tube, 0.3 µL of Lipofectamine 3000 was diluted and well mixed into 10 µL of Opti-MEM I reduced serum medium (Gibco). In the other tube, 10 µL of Opti-MEM I was first mixed with 0.45 µL of the P3000 reagent by vertexing and then mixed with 45 ng of plasmid DNA (up to 1.75 µL volume) by flicking. The two tubes were then combined, mixed by a brief vertex, and incubated at room temperature for 10 min. For each plasmid, 5 µL of the above DNA/Lipofectamine complex was added to each well for a total of four wells. The amount of plasmid DNA was equivalent to 10 ng for each well, which was determined to be optimal in a separate titration experiment (Fig. p values for all data figures are provided in Additional file The raw luminescence readouts were processed in Excel in a stepwise manner. First, the Fluc signal was normalized to the corresponding Rluc signal for each well. Second, the average Fluc/Rluc ratio for the no-promoter vector, pLK037, was calculated from its four replicate wells. Third, the Fluc/Rluc ratio of each well was divided by the average pLK037 ratio from step 2 above. This step effectively sets the average Fluc/Rluc ratio of pLK037 to 1, which represents the assay background. Lastly, the normalized promoter activity for each promoter construct was calculated as the average of the normalized Fluc/Rluc ratios among the four replicate wells. The corresponding standard error was calculated as the standard deviation divided by the square root of the number of replicates. Statistical comparison between any two promoter constructs was performed in RStudio using the pairwise.t.test function with Benjamini–Hochberg correction for multiple testing (adjusted Additional file 1: Promoter constructs and corresponding sequences. Table S1. Promoters assayed in Figure 2. Table S2. Promoters assayed in Figure 3. Table S3, Promoters assayed in Figure 4. Table S4. Copy number of two-monomer promoters and individual monomer and tether domains in the mouse genome.Additional file 2: F9 data, sequence alignments and cell line authentication report. Figure S1. Comparison of sense and antisense promoter activities for two-monomer mouse L1 5’UTR consensus sequences in F9 cells. Figure S2. Differential contribution of monomer 2, monomer 1 and tether to overall promoter activity in F9 cells. Figure S3. Contribution of different lengths of monomer 3 to overall promoter activity in F9 cells. Figure S4. Alignment of M2 from A_I, Gf_I and Tf_I subfamilies. Figure S5. Alignment of A_I monomers. Figure S6. Alignment of Tf_I monomers. Figure S7. Alignment of Gf_I monomers. Figure S8. Alignment of tether sequences. Figure S9. Cell line authentication report for NIH/3T3 subline used.Additional file 3: Adjusted p values from pairwise t-tests with Benjamini-Hochberg correction for promoter constructs in all data figures.